Folate Receptor 1 Antibodies and Immunoconjugates and Uses Thereof

ABSTRACT

Novel anti-cancer agents, including, but not limited to, antibodies and immunoconjugates, that bind to human folate receptor 1 are provided. Methods of using the agents, antibodies, or immunoconjugates, such as methods of inhibiting tumor growth are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/800,835, filed Mar. 13, 2013, now U.S. Pat. No. 9,133,275, issuedSep. 15, 2015, which is a divisional application of U.S. applicationSer. No. 13/033,723, filed Feb. 24, 2011, now U.S. Pat. No. 8,557,966,issued Oct. 15, 2013, which claims the priority benefit of U.S.Provisional Application No. 61/307,797, filed Feb. 24, 2010, U.S.Provisional Application No. 61/346,595, filed May 20, 2010, and U.S.Provisional Application No. 61/413,172, filed Nov. 12, 2010, each ofwhich is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The field of this invention generally relates to antibodies andimmunoconjugates that bind to human folate receptor 1, as well as tomethods of using the antibodies and immunoconjugates for the treatmentof diseases, such as cancer.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich—breast, lung, colorectal, and prostate—account for over half ofall new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

Folate Receptor 1 (FOLR1), also known as Folate Receptor-alpha, orFolate Binding Protein, is an N-glycosylated protein expressed on plasmamembrane of cells. FOLR1 has a high affinity for folic acid and forseveral reduced folic acid derivatives. FOLR1 mediates delivery of thephysiological folate, 5-methyltetrahydrofolate, to the interior ofcells.

FOLR1 is overexpressed in vast majority of ovarian cancers, as well asin many uterine, endometrial, pancreatic, renal, lung, and breastcancers, while the expression of FOLR1 on normal tissues is restrictedto the apical membrane of epithelial cells in the kidney proximaltubules, alveolar pneumocytes of the lung, bladder, testes, choroidplexus, and thyroid (Weitman S D, et al., Cancer Res 52: 3396-3401(1992); Antony A C, Annu Rev Nutr 16: 501-521 (1996); Kalli K R, et al.Gynecol Oncol 108: 619-626 (2008)). This expression pattern of FOLR1makes it a desirable target for FOLR1-directed cancer therapy.

Because ovarian cancer is typically asymptomatic until advanced stage,it is often diagnosed at a late stage and has poor prognosis whentreated with currently available procedures, typically chemotherapeuticdrugs after surgical de-bulking (von Gruenigen V et al., Cancer 112:2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81 e81-86(2007); Harry V N et al., Obstet Gynecol Surv 64: 548-560 (2009)). Thusthere is a clear unmet medical need for more effective therapeutics forovarian cancers.

Three anti-FOLR1 antibodies have been examined as potential anti-cancerdrugs. Murine monoclonal antibodies Mov18 and Mov19 were isolated in thelate 1980s (Miotti S et al., Int J Cancer 39: 297-303 (1987)), confirmedto target FOLR1 (Coney L R et al., Cancer Res 51: 6125-6132 (1991)), andtested in pre-clinical studies for their ability to eradicateantigen-expressing cancer cells as conjugates with a cytotoxicribosome-inactivating protein (Conde F P et al., Eur J Biochem 178:795-802 (1989)).

Mov19 was tested as a bi-specific antibody targeting cytotoxic T cellsand natural killer cells (Mezzanzanica D et al., Int J Cancer 41:609-615 (1988); Ferrini S et al., Int J Cancer Suppl 4: 53-55 (1989);Ferrini S et al., Int J Cancer 48: 227-233 (1991)), and as a fusionprotein of the single-chain Fv (scFv) of Mov19 with interleukin-2 invivo (Melani C et al., Cancer Res 58: 4146-4154 (1998)). Chimeric(murine variable/human constant) anti-FOLR1 antibodies Mov18 and Mov19have been examined pre-clinically on their ability to mediate cytotoxicimmune cell-dependent killing of FOLR1-expressing tumor cells in vitro(Coney L R et al., Cancer Res 54: 2448-2455 (1994)), and a chimericMov18-IgE was tested in IgE-dependent immunotherapeutic preclinicalmodels (Karagiannis S N et al., J Immunol 179: 2832-2843 (2007); Gould HJ et al., Eur J Immunol 29: 3527-3537 (1999)).

Mov18 was studied in the form of conjugates with various radionuclidesin preclinical studies and then, in early 1990s, in clinical trials(Zacchetti A et al., Nucl Med Biol 36: 759-770 (2009)), which endedwithout any drug being approved for clinical use.

MORAb003, a humanized form of the murine monoclonal anti-FOLR1 antibodyLK26 was evaluated pre-clinically as a non-modified antibody (Ebel W etal., Cancer Immun 7:6 (2007)) and as a conjugate with the ¹¹¹Inradionuclide (Smith-Jones P M et al., Nucl Med Biol 35: 343-351 (2008)),and is currently undergoing clinical trials as a non-modified antibody(D. K. Armstrong et al. J. Clin. Oncol. 26: 2008, May 20 suppl; abstract5500).

SUMMARY OF THE INVENTION

The present invention provides novel antibodies that bind to humanfolate receptor 1, immunoconjugates comprising these antibodies, andmethods of their use. The present invention further provides novelpolypeptides, such as antibodies that bind human folate receptor 1,fragments of such antibodies, and other polypeptides related to suchantibodies. Polynucleotides comprising nucleic acid sequences encodingthe polypeptides are also provided, as are vectors comprising thepolynucleotides. Cells comprising the polypeptides and/orpolynucleotides of the invention are further provided. Compositions(e.g., pharmaceutical compositions) comprising the novel folate receptor1 antibodies or immunoconjugates are also provided. In addition, methodsof making and using the novel folate receptor 1 antibodies orimmunoconjugates are also provided, such as methods of using the novelfolate receptor 1 antibodies or immunoconjugates to inhibit tumor growthand/or treat cancer.

Thus, in one aspect, the invention provides a humanized antibody orantigen binding fragment thereof that specifically binds a human folatereceptor 1, wherein the antibody comprises (a) a heavy chain CDR1comprising GYFMN (SEQ ID NO:1); a heavy chain CDR2 comprisingRIHPYDGDTFYNQXaa₁FXaa₂Xaa₃ (SEQ ID NO:56); and a heavy chain CDR3comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprisingRASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQID NO:9); wherein Xaa₁ is selected from K, Q, H, and R; Xaa₂ is selectedfrom Q, H, N, and R; and Xaa₃ is selected from G, E, T, S, A, and V. Ina certain embodiment, the humanized antibody or antigen binding fragmentthereof binds a human folate receptor 1 with substantially the sameaffinity as the antibody chimeric Mov19. In a certain embodiment, thehumanized antibody or antigen binding fragment thereof comprises theheavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQ ID NO:2).

In a certain embodiment, the binding affinity is measured by flowcytometry, Biacore, or radioimmunoassay.

In another embodiment, the invention provides a humanized antibody orantigen binding fragment thereof that specifically binds a human folatereceptor 1, wherein the antibody comprises: (a) a heavy chain CDR1comprising GYFMN (SEQ ID NO:1), or a variant thereof comprising 1, 2, 3,or 4 conservative amino acid substitutions; a heavy chain CDR2comprising RIHPYDGIDTFYNQKFQG (SEQ ID NO:2), or a variant thereofcomprising 1, 2, 3, or 4 amino conservative acid substitutions; and aheavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions;and/or (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:7),or a variant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; a light chain CDR2 comprising RASNLEA (SEQ ID NO:8), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and a light chain CDR3 comprising QQSREYPYT (SEQ IDNO:9), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions.

In a certain embodiment, the invention provides a humanized antibody orantigen binding fragment thereof that specifically binds the humanfolate receptor 1 comprising the heavy chain of SEQ ID NO:6. In anotherembodiment, the humanized antibody or antigen binding fragment thereofis encoded by the plasmid DNA deposited with the ATCC on Apr. 7, 2010and having ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.

In a certain embodiment, the invention provides a humanized antibody orantigen binding fragment thereof that competes for binding to FOLR1 withan antibody comprising (a) a heavy chain CDR1 comprising GYFMN (SEQ IDNO:1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa₁FXaa₂Xaa₃ (SEQ IDNO:56); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:3); and(b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a lightchain CDR2 comprising RASNLEA (SEQ ID NO:8); and a light chain CDR3comprising QQSREYPYT (SEQ ID NO:9); wherein Xaa₁ is selected from K, Q,H, and R; Xaa₂ is selected from Q, H, N, and R; and Xaa₃ is selectedfrom G, E, T, S, A, and V. In a certain embodiment, the humanizedantibody comprises the heavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQID NO:2).

In a certain embodiment, the invention provides a polypeptide, humanizedantibody or antigen binding fragment thereof comprising a heavy chainvariable domain at least about 90% identical to SEQ ID NO:4, and a lightchain variable domain at least about 90% identical to SEQ ID NO:10 orSEQ ID NO:11. In another embodiment, the humanized antibody or antigenbinding fragment comprises a heavy chain variable domain at least about95% identical to SEQ ID NO:4, and a light chain variable domain at leastabout 95% identical to SEQ ID NO:10 or SEQ ID NO: 11. In a furtherembodiment, the humanized antibody comprises a heavy chain variabledomain at least about 99% identical to SEQ ID NO:4, and a light chainvariable domain at least about 99% identical to SEQ ID NO: 10 or SEQ IDNO: 11. In a certain embodiment, the humanized antibody comprises theheavy chain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO:10 or SEQ ID NO: 11. In certain embodiments, theinvention provides a polypeptide, antibody, or antigen binding fragmentat least about 90% identical to SEQ ID NOs: 88-119. In certainembodiments, the invention provides a polypeptide, antibody, or antigenbinding fragment at least about 95% identical to SEQ ID NOs: 88-119. Incertain embodiments, the invention provides a polypeptide, antibody, orantigen binding fragment at least about 99% identical to SEQ ID NOs:88-119.

In a certain embodiment, the invention provides a humanized antibody orantigen binding fragment thereof that is expressed at least ten-foldhigher than chMov19 in eukaryotic cells. In a certain embodiment, theeukaryotic cells are HEK-293T cells.

In certain embodiments, the invention provides an antibody or antigenbinding fragment thereof that specifically binds a human folate receptor1, wherein the antibody comprises:

(a) a heavy chain CDR1 comprising SSYGMS (SEQ ID NO:30); a heavy chainCDR2 comprising TISSGGSYTY (SEQ ID NO:31); and/or a heavy chain CDR3comprising DGEGGLYAMDY (SEQ ID NO:32); and/or (b) a light chain CDR1comprising KASDHINNWLA (SEQ ID NO:27); a light chain CDR2 comprisingGATSLET (SEQ ID NO:28); and a light chain CDR3 comprising QQYWSTPFT (SEQID NO:29). In another embodiment, the invention provides an antibody orantigen binding fragment thereof that specifically binds a human folatereceptor 1, wherein the antibody comprises: (a) a heavy chain CDR1comprising TNYWMQ (SEQ ID NO:60); a heavy chain CDR2 comprisingAIYPGNGDSR (SEQ ID NO:61); and/or a heavy chain CDR3 comprising RDGNYAAY(SEQ ID NO:62); and/or (b) a light chain CDR1 comprising RASENIYSNLA(SEQ ID NO:57); a light chain CDR2 comprising AATNLAD (SEQ ID NO:58);and a light chain CDR3 comprising QHFWASPYT (SEQ ID NO:59). In anotherembodiment, the invention provides an antibody or antigen bindingfragment thereof that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMY(SEQ ID NO:66); a heavy chain CDR2 comprising AIYPGNSDTT (SEQ ID NO:67);and/or a heavy chain CDR3 comprising RHDYGAMDY (SEQ ID NO:68); and/or(b) a light chain CDR1 comprising RASENIYTNLA (SEQ ID NO:63); a lightchain CDR2 comprising TASNLAD (SEQ ID NO:64); and a light chain CDR3comprising QHFWVSPYT (SEQ ID NO:65). In another embodiment, theinvention provides an antibody or antigen binding fragment thereof thatspecifically binds a human folate receptor 1, wherein the antibodycomprises: (a) a heavy chain CDR1 comprising SSFGMH (SEQ ID NO:72); aheavy chain CDR2 comprising YISSGSSTIS (SEQ ID NO:73); and/or a heavychain CDR3 comprising EAYGSSMEY (SEQ ID NO:74); and/or (b) a light chainCDR1 comprising RASQNINNNLH (SEQ ID NO:69); a light chain CDR2comprising YVSQSVS (SEQ ID NO:70); and a light chain CDR3 comprisingQQSNSWPHYT (SEQ ID NO:71). In another embodiment, the invention providesan antibody or antigen binding fragment thereof that specifically bindsa human folate receptor 1, wherein the antibody comprises: (a) a heavychain CDR1 comprising TSYTMH (SEQ ID NO:78); a heavy chain CDR2comprising YINPISGYTN (SEQ ID NO:79); and/or a heavy chain CDR3comprising GGAYGRKPMDY (SEQ ID NO:80); and/or (b) a light chain CDR1comprising KASQNVGPNVA (SEQ ID NO:75); a light chain CDR2 comprisingSASYRYS (SEQ ID NO:76); and a light chain CDR3 comprising QQYNSYPYT (SEQID NO:77).

In certain embodiments, the polypeptides of the invention arefull-length antibodies or antigen binding fragments. In certainembodiments, the antibodies or antigen binding fragments are a Fab, aFab′, a F(ab′)2, a Fd, a single chain Fv or scFv, a disulfide linked Fv,a V NAR domain, a IgNar, an intrabody, an IgG-CH2, a minibody, aF(ab′)3, a tetrabody, a triabody, a diabody, a single-domain antibody,DVD-Ig, Fcab, mAb2, a (scFv)2, or a scFv-Fc.

In certain embodiments, an antibody or polypeptide of the inventionbinds to a human folate receptor 1 with a Kd of about 1.0 to about 10nM. In one embodiment, the antibody or polypeptide binds to a humanfolate receptor 1 with a Kd of about 1.0 nM or better. In a certainembodiment, binding affinity is measured by flow cytometry, Biacore, orradioimmunoassay.

The invention also provides a method of making an antibody of theinvention comprising culturing a cell expressing said antibody; and (b)isolating the antibody from said cultured cell. In a certain embodiment,the cell is a eukaryotic cell.

The invention also provides an immunoconjugate having the formula(A)-(L)-(C), wherein: (A) is an antibody or antigen binding fragment orpolypeptide of the invention; (L) is a linker; and (C) is a cytotoxicagent, wherein said linker (L) links (A) to (C).

In one embodiment, the linker is selected from the group of a cleavablelinker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylicacid based linker. In a further embodiment, the linker is selected fromthe group consisting: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP)or N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP);N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC);N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide). In a certain embodiment, the linker isN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide).

In one embodiment, the immunoconjugates comprise a cytotoxic agentselected from the group of a maytansinoid, maytansinoid analog,benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin,duocarmycin analog, calicheamicin, dolastatin, dolastatin analog,auristatin, tomaymycin derivative, and leptomycin derivative or aprodrug of the agent. In a further embodiment, the cytotoxic agent is amaytansinoid. In another embodiment, the cytotoxic agent isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine.

In one embodiment the invention provides an immunoconjugate comprising:(A) a humanized antibody comprising the heavy chain variable domain ofSEQ ID NO:4, and the light chain variable domain of SEQ ID NO:10 or SEQID NO:11; (L)N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide); and (C)N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein(L) links (A) to (C).

In one embodiment the invention provides an immunoconjugate comprising:(A) a humanized antibody comprising the heavy chain variable domain ofSEQ ID NO:4, and the light chain variable domain of SEQ ID NO: 10 or SEQID NO: 11; (L) N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); and(C) N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine;wherein (L) links (A) to (C).

In one embodiment the invention provides an immunoconjugate comprising:(A) a humanized antibody comprising the heavy chain variable domain ofSEQ ID NO:4, and the light chain variable domain of SEQ ID NO:10 or SEQID NO:11; (L) N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate(sulfo-SPDB); and (C)N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein(L) links (A) to (C).

In one embodiment the invention provides an immunoconjugate comprising:(A) a humanized antibody comprising the heavy chain variable domain ofSEQ ID NO:4, and the light chain variable domain of SEQ ID NO:10 or SEQID NO: 11; (L) N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate(sulfo-SPP); and (C)N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)links (A) to (C).

In one embodiment the invention provides an immunoconjugate comprising:(A) a humanized antibody comprising the heavy chain variable domain ofSEQ ID NO:4, and the light chain variable domain of SEQ ID NO:10 or SEQID NO:11; (L) N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); and(C) N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine; wherein(L) links (A) to (C).

The invention also provides a pharmaceutical composition comprising anantibody, antigen binding fragment, polypeptide, or immunoconjugate ofthe invention and a pharmaceutically acceptable carrier. In a certainembodiment, the pharmaceutical composition further comprises a secondanti-cancer agent.

The invention also provides a diagnostic reagent comprising an antibody,antigen binding fragment, polypeptide, or immunoconjugate of theinvention which is labeled. In one embodiment, the label is selectedfrom the group of a radiolabel, a fluorophore, a chromophore, an imagingagent and a metal ion.

The invention also provides a kit comprising the antibody, antigenbinding fragment, polypeptide, or immunoconjugate of the invention.

The invention also provides a method of inhibiting tumor growth in asubject, comprising administering a therapeutically effective amount ofthe antibody, antigen binding fragment, polypeptide, immunoconjugate, orpharmaceutical composition of the invention to the subject. In a certainembodiment, the invention provides a method of inhibiting tumor growthin a subject comprising administering a therapeutically effective amountof an immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is anantibody or antigen binding fragment thereof that specifically binds ahuman folate receptor 1; (L) is a linker; and (C) is a cytotoxinselected from the group consisting of a maytansinoid and a maytansinoidanalog; wherein (L) links (A) to (C) and wherein the immunoconjugatereduces mean tumor volume at least two-fold in a KB xenograft model. Ina certain embodiment, the method comprises administering an antibody orantigen binding fragment thereof that comprises (a) a heavy chain CDR1comprising GYFMN (SEQ ID NO:1); a heavy chain CDR2 comprisingRIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO:56); and a heavy chain CDR3comprising YDGSRAMDY (SEQ ID NO:3); and (b) a light chain CDR1comprising KASQSVSFAGTSLMH (SEQ ID NO:7); a light chain CDR2 comprisingRASNLEA (SEQ ID NO:8); and a light chain CDR3 comprising QQSREYPYT (SEQID NO:9); wherein Xaa₁ is selected from K, Q, H, and R; Xaa₂ is selectedfrom Q, H, N, and R; and Xaa₃ is selected from G, E, T, S, A, and V. Ina further embodiment, the antibody comprises a heavy chain CDR2comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:2).

In a certain embodiment, the invention provides a method for inhibitingtumor growth comprising administering an antibody or antigen bindingfragment thereof encoded by the plasmid DNA deposited with the ATCC onApr. 7, 2010 and having ATCC deposit nos. PTA-10772 and PTA-10773 or10774.

In another embodiment, the method provides administering animmunoconjugate comprising a humanized antibody comprising the heavychain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO:10 or SEQ ID NO:11; (L)N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide); and (C)N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine.

In another embodiment, the method comprises administering animmunoconjugate which comprises (A) a humanized antibody comprising theheavy chain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB); and (C)N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein(L) links (A) to (C).

In another embodiment, the method comprises administering animmunoconjugate which comprises (A) a humanized antibody comprising theheavy chain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB); and (C)N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine; wherein(L) links (A) to (C).

In another embodiment, the method comprises administering animmunoconjugate which comprises (A) a humanized antibody comprising theheavy chain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP); and (C)N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)links (A) to (C).

In another embodiment, the method comprises administering animmunoconjugate which comprises (A) a humanized antibody comprising theheavy chain variable domain of SEQ ID NO:4, and the light chain variabledomain of SEQ ID NO:10 or SEQ ID NO:11; (L) N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP); and (C)N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine; wherein (L)links (A) to (C).

In another embodiment, the method comprises administering animmunoconjugate which comprises the antibody huFR-1-21 deposited withATCC on Apr. 7, 2010 and having ATCC deposit nos. PTA-10775 andPTA-10776. In a certain embodiment, the huFR1-21 antibody comprises (a)a heavy chain CDR1 comprising SSYGMS (SEQ ID NO:30); a heavy chain CDR2comprising TISSGGSYTY (SEQ ID NO:31); and a heavy chain CDR3 comprisingDGEGGLYAMDY (SEQ ID NO:32); and (b) a light chain CDR1 comprisingKASDHINNWLA (SEQ ID NO:27); a light chain CDR2 comprising GATSLET (SEQID NO:28); and a light chain CDR3 comprising QQYWSTPFT (SEQ ID NO:29).In certain embodiments the method comprises administering animmunoconjugate which comprises the antibody is the huFR1-48 antibodywhich comprises: (a) a heavy chain CDR1 comprising TNYWMQ (SEQ IDNO:60); a heavy chain CDR2 comprising AIYPGNGDSR (SEQ ID NO:61); and aheavy chain CDR3 comprising RDGNYAAY (SEQ ID NO:62); and (b) a lightchain CDR1 comprising RASENIYSNLA (SEQ ID NO:57); a light chain CDR2comprising AATNLAD (SEQ ID NO:58); and a light chain CDR3 comprisingQHFWASPYT (SEQ ID NO:59). In certain embodiments the method comprisesadministering an immunoconjugate which comprises the antibody is thehuFR1-49 antibody which comprises: (a) a heavy chain CDR1 comprisingTNYWMY (SEQ ID NO:66); a heavy chain CDR2 comprising AIYPGNSDTT (SEQ IDNO:67); and a heavy chain CDR3 comprising RHDYGAMDY (SEQ ID NO:68); and(b) a light chain CDR1 comprising RASENIYTNLA (SEQ ID NO:63); a lightchain CDR2 comprising TASNLAD (SEQ ID NO:64); and a light chain CDR3comprising QHFWVSPYT (SEQ ID NO:65). In certain embodiments the methodcomprises administering an immunoconjugate which comprises the antibodyis the huFR1-57 antibody which comprises: (a) a heavy chain CDR1comprising SSFGMH (SEQ ID NO:72); a heavy chain CDR2 comprisingYISSGSSTIS (SEQ ID NO:73); and a heavy chain CDR3 comprising EAYGSSMEY(SEQ ID NO:74); and (b) a light chain CDR1 comprising RASQNINNNLH (SEQID NO:69); a light chain CDR2 comprising YVSQSVS (SEQ ID NO:70); and alight chain CDR3 comprising QQSNSWPHYT (SEQ ID NO:71). In certainembodiments the method comprises administering an immunoconjugate whichcomprises the antibody is the huFR1-65 antibody which comprises: (a) aheavy chain CDR1 comprising TSYTMH (SEQ ID NO:78); a heavy chain CDR2comprising YINPISGYTN (SEQ ID NO:79); and a heavy chain CDR3 comprisingGGAYGRKPMDY (SEQ ID NO:80); and (b) a light chain CDR1 comprisingKASQNVGPNVA (SEQ ID NO:75); a light chain CDR2 comprising SASYRYS (SEQID NO:76); and a light chain CDR3 comprising QQYNSYPYT (SEQ ID NO:77).

In one embodiment, the method inhibits ovarian tumor, brain tumor,breast tumor, uterine tumor, endometrial tumor, pancreatic tumor, renaltumor, or lung tumor growth. In a certain embodiment, the methodinhibits ovarian tumor growth. In another embodiment, the inventioninhibits lung tumor growth. In a certain embodiment, tumor growthinhibition is used to treat cancer. In a further embodiment, the methodcomprises administering a second anti-cancer agent to the subject. In acertain embodiment, the second anti-cancer agent is a chemotherapeuticagent.

The invention also provides an isolated cell producing the antibody,antigen binding fragment, or polypeptide of the invention.

The invention also provides an isolated polynucleotide comprising asequence at least 90% identical to a sequence selected from the groupconsisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48, and120-127. In a certain embodiment, the isolated polynucleotide is atleast 95% identical a sequence selected from the group consisting of SEQID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48, and 120-127. In anotherembodiment, isolated polynucleotide is at least 99% identical to asequence selected from the group consisting of SEQ ID NOs: 5, 14, 15,37, 38, 43, 44, 47, 48, and 120-127. The invention also provides avector comprising any of the polynucleotides of SEQ ID NOs: 5, 14, 15,37, 38, 43, 44, 47, 48, and 120-127. In another embodiment, theinvention provides a host cell comprising a vector which contains apolynucleotide of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48, and120-127.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D. Surface residues for murine (muMov19) andhumanized (huMov19) Mov19. (FIG. 1A) Murine and humanized Mov19 lightchain surface residues. The murine and humanized Mov19 light chainvariable region frame surface residues and position number (Kabatsystem) are given. The human residues that are different from theoriginal murine sequences are underlined. *Position 74 is not a surfaceposition, but to remove a consensus N-linked glycosylation site inversion 1.00, this position was changed to a Threonine (the most commonhuman residue in this position), resulting in version 1.60. (FIG. 1B)Murine and Human Mov19 Heavy Chain Surface Residues. The murine andhumanized Mov19 heavy chain variable region frame surface residues andposition number (Kabat system) are given. The human residues that aredifferent from the original murine sequences are underlined. Similarsurface residues are provided for FR1-21 (FIGS. 1C and 1D).

FIG. 2. Alignments of chimeric Mov19 and huMov19 heavy and light chainvariable domains and muFR1-21 and huFR-21 heavy and light clain variabledomains. Alignment of resurfaced sequences for the Mov19 and Fr1-21variable regions with their murine counterparts. (FIG. 2, Panel A) and(FIG. 2, Panel C) light chain variable domains; (FIG. 2, Panel B) and(FIG. 2, Panel D) heavy chain variable domain. Dashes “-” denoteidentity with the murine sequence. The CDRs (Kabat definition) areunderlined.

FIG. 3. Expression of chimeric Mov19 and huMov19 in HEK cells. Thechimeric and human Mov19 expression plasmids were transientlytransfected into suspension HEK293-T cells, harvested 7 days later, andthe expressed antibody was determined by quantitative ELISA. The lightchain and heavy chain plasmids were transfected at either 3:1 or 6:1respective molar ratios.

FIG. 4. Binding specificity of anti-FOLR1 antibodies, as detected bytheir binding to FOLR1-expressing 300-19 cells. The binding of huMov19to 300-19-FOLR1 cells by flow cytometry. 300-19 parental cellsexpressing FOLR-1. The grey solid shading represents cellular autofluorescence; the black dotted lines represent cells incubated withanti-human secondary antibody conjugated with FITC, the black solidlines represent cells incubated with the huMov-19 antibody andanti-human secondary antibody conjugated to FITC.

FIG. 5. Binding affinities and in vitro cytotoxic activity of anti-FOLR1antibodies and immunoconjugates. Binding affinity of huMov19 and variousmurine and humanized FR-1 antibodies was measured on SKOV3 cells. Invitro cytotoxic activity of PEG4-Mal-DM4 conjugates of the listedantibodies was also assayed.

FIG. 6. Antibody-dependent cellular cytotoxicity of immunoconjugates.ADCC activity of huMov19, huFR1-21, and Mor003 was assayed againstIgrov1 cells. Igrov 1 were incubated at 15000 cells/well Target:NK cellratio of 1:4.

FIG. 7. Cytoxic activity of continuous exposure of huFR1-21-PEG4-mal-DM4and huMov19-PEG4-mal-DM4 on KB cells. An excess of non-conjugatedantibodies suppressed the activity of immunoconjugates when they wereco-incubated in the presence of KB cells, indicating cytotoxic activityis antigen-dependent.

FIG. 8. In vivo efficacy of huMov19-targeted conjugates in a KBxenograft model. FOLR1-targeting cleavable conjugate huMov19-SPDB-DM4(B) in comparison with non-FOLR1-targeting huC242-SPDB-DM4 (D), andnon-cleavable conjugate huMov19-PEG4-Mal-DM4 (C) in comparison withnon-targeting huC242-PEG4Mal-DM4 (E) were tested using an establishedxenograft model of KB cells implanted subcutaneous into SCID mice.Targeting of FOLR1 by huMov19 resulted in significant reduction in meantumor volume.

FIG. 9. In vivo efficacy of huMov19-PEG4-Mal-DM4 compared to murine FR-1anti-FOLR1 antibodies in a KB xenograft model. FR-1 series antibodies,either unconjugated, or conjugated with PEG4-Mal-DM4 were tested fortheir ability to reduce mean tumor volume compared tohuMov19-PEG4-Mal-DM4 in a KB xenograft tumor model. (FIG. 9, Panel A)FR-1-9, (FIG. 9, Panel B) FR-1-13, (FIG. 9, Panel C) FR-1-22, and (FIG.9, Panel D) FR-1-23.

FIG. 10. In vivo efficacy of huMov19-PEG4-Mal-DM4 andhuFR1-21-PEG4-Mal-DM4 in a KB xenograft model. 10 mg/kg singleinjections of huMov19-PEG4-Mal-DM4 and huFR1-21-PEG4-Mal-DM4 on day 6post inoculation was performed. Both huMov19-PEG4-Mal-DM4 andhuFR1-21-PEG4-Mal-DM4 showed a significant reduction in mean tumorvolume. “Mean TV” refers to mean tumor volume.

FIG. 11. HuMov19-PEG4-mal-DM4 shows dose dependent activity in the KBxenograft model. Dose dependent activity of the immunoconjugate wasassayed across the range of doses tested. Weekly dosing resulted inimprovement of anti-tumor activity. High drug loads only marginallyimproved activity in the 10 mg/kg dose groups, with reduced activity inthe lower dose groups. 3.7 DAR refers to 3.7 drug molecules perantibody.

FIG. 12. In vivo efficacy of huMov19 conjugated with DM1 and DM4 withvarious linkers. huMov19 was conjugated to SMCC-DM1 at 3.9 drugmolecules per antibody (FIG. 12, Panel A), sulfo-mal-DM4 at 3.7 drugmolecules per antibody (FIG. 12, Panel B), and sulfo-mal-DM4 at 8.23drug molecules per antibody (FIG. 12, Panel C) and assayed for theirability to reduce mean tumor volume at various concentrations comparedto huMov19-PEG4-mal-DM4.

FIG. 13. In vivo efficacy of huMov19 conjugated with DM1 and DM4 withvarious linkers. huMov19 was conjugated to SPP-DM1 at 4.3 drug moleculesper antibody; sulfo-SPDB-DM4 at 3.8 drug molecules per antibody,SPDB-DM4 at 3.8 drug molecules per antibody, and sulfo-SPDB-DM4 at 6.8drug molecules per antibody and assayed for their ability to reduce meantumor volume. Mice were treated with 5 mg/kg (FIG. 13, Panel A) and 2.5mg/kg (FIG. 13, Panel B) of one of the conjugates listed above or withPBS only.

FIG. 14. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in OVCAR-3 xenografttumor model. Mice were treated with 25, 50, or 100 μg/kg ofhuMov19-sulfo-SPDB-DM4 or with PBS only.

FIG. 15. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in IGROV-1 xenografttumor model. Mice were treated with 25, 50, or 100 μg/kg ofhuMov19-sulfo-SPDB-DM4 or with PBS only

FIG. 16. In vivo efficacy of huMov19-sulfo-SPDB-DM4 in OV-90 xenografttumor model. Mice were treated with 25, 50, or 100 μg/kg ofhuMov19-sulfo-SPDB-DM4 or with PBS only.

FIG. 17. Effect of cleavable and non-cleavable linkers on efficacy ofimmunoconjugates in KB xenograft models.

FIG. 18. Effect of cleavable linkers on efficacy of immunoconjugates in(FIG. 18, Panel A) KB xenograft model (FIG. 18, Panel B) OVCAR-3xenograft model.

FIG. 19. In vitro and in vivo efficacy of huFR1-48, huFR1-49, huFR1-57,and huFR1-65-SMCC-DM1 in KB and xenograft tumor models. Mice weretreated with 200 μg/kg single doses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto polypeptides such as antibodies, and immunoconjugates that bind tohuman folate receptor 1 (FOLR1). Related polypeptides andpolynucleotides, compositions comprising the FOLR1-binding agents, andmethods of making the FOLR1-binding agents are also provided. Methods ofusing the novel FOLR1-binding agents, such as methods of inhibitingtumor growth and/or treating cancer, are further provided.

I. DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “human folate receptor 1” or “FOLR1”, as used herein, refersto any native human FOLR1, unless otherwise indicated. The term “FOLR1”encompasses “full-length,” unprocessed FOLR1 as well as any form ofFOLR1 that results from processing within the cell. The term alsoencompasses naturally occurring variants of FOLR1, e.g., splicevariants, allelic variants and isoforms. The FOLR1 polypeptidesdescribed herein can be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods. Examples of FOLR1 sequences include, but are notlimited to NCBI reference numbers P15328, NP_(—)001092242.1, AAX29268.1,AAX37119.1, NP_(—)057937.1, and NP_(—)057936.1.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as FOLR1.In a certain embodiment blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. Desirably, the biological activity is reduced by 10%, 20%, 30%,50%, 70%, 80%, 90%, 95%, or even 100%.

The term “anti-FOLR1 antibody” or “an antibody that binds to FOLR1”refers to an antibody that is capable of binding FOLR1 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FOLR1. The extent of binding of ananti-FOLR1 antibody to an unrelated, non-FOLR1 protein is less thanabout 10% of the binding of the antibody to FOLR1 as measured, e.g., bya radioimmunoassay (RIA). In certain embodiments, an antibody that bindsto FOLR1 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1nM, or ≦0.1 nM.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539 or5,639,641.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-Iazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better”, the antibody's affinity for theantigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicsmeasured by said values (e.g., Kd values). The difference between saidtwo values is less than about 50%, less than about 40%, less than about30%, less than about 20%, or less than about 10% as a function of thevalue for the reference/comparator antibody.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is linked to a cell binding agent(i.e., an anti-FOLR1 antibody or fragment thereof) and is defined by ageneric formula: C-L-A, wherein C=cytotoxin, L=linker, and A=cellbinding agent or anti-FOLR1 antibody or antibody fragment.Immunoconjugates can also be defined by the generic formula in reverseorder: A-L-C.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a maytansinoid, to a cell-binding agent such asan anti FOLR1 antibody or a fragment thereof in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude charged linkers, and hydrophilic forms thereof as describedherein and know in the art.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancers.

“Tumor” and “neoplasm” refer to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulation can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and in a certainembodiment, stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and in a certain embodiment, stop)tumor metastasis; inhibit, to some extent, tumor growth; and/or relieveto some extent one or more of the symptoms associated with the cancer.See the definition herein of “treating”. To the extent the drug canprevent growth and/or kill existing cancer cells, it can be cytostaticand/or cytotoxic. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically but not necessarily,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkyatingagents, antimetabolites, spindle poison plant alkaloids,cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars can be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, orcan be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls can also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages can be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

The term “vector” means a construct, which is capable of delivering, andexpressing, one or more gene(s) or sequence(s) of interest in a hostcell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTcan be used as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In certain embodiments, identity exists over a region of the sequencesthat is at least about 10, about 20, about 40-60 residues in length orany integral value therebetween, or over a longer region than 60-80residues, at least about 90-100 residues, or the sequences aresubstantially identical over the full length of the sequences beingcompared, such as the coding region of a nucleotide sequence forexample.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the FOLR1 to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

II. FOLR1-BINDING AGENTS

The present invention provides agents that specifically bind humanFOLR1. These agents are referred to herein as “FOLR1-binding agents.”The full-length amino acid (aa) and nucleotide (nt) sequences for FOLR1are known in the art and also provided herein as represented by SEQ IDNOs:25 and 26, respectively.

In certain embodiments, the FOLR1 binding agents are antibodies,immunoconjugates or polypeptides. In some embodiments, the FOLR1 bindingagents are humanized antibodies. In certain embodiments, the FOLR-1binding agents are humanized versions of the murine Mov19 antibody(variable heavy and light chain shown as SEQ ID NOs: 17 and 18respectively).

In certain embodiments, the FOLR1-binding agents have one or more of thefollowing effects: inhibit proliferation of tumor cells, reduce thetumorigenicity of a tumor by reducing the frequency of cancer stem cellsin the tumor, inhibit tumor growth, increase survival, trigger celldeath of tumor cells, differentiate tumorigenic cells to anon-tumorigenic state, or prevent metastasis of tumor cells.

In certain embodiments, immunoconjugates or other agents thatspecifically bind human FOLR1 trigger cell death via a cytotoxic agent.For example, in certain embodiments, an antibody to a human FOLR1antibody is conjugated to a maytansinoid that is activated in tumorcells expressing the FOLR1 by protein internalization. In certainalternative embodiments, the agent or antibody is not conjugated.

In certain embodiments, the FOLR1-binding agents are capable ofinhibiting tumor growth. In certain embodiments, the FOLR1-bindingagents are capable of inhibiting tumor growth in vivo (e.g., in axenograft mouse model and/or in a human having cancer). In certainembodiments, the FOLR1-binding agents are capable of inhibiting tumorgrowth in a human.

Thus, the invention provides a humanized antibody or antigen bindingfragment thereof that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN(SEQ ID NO:1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa₁FXaa₂Xaa₃(SEQ ID NO:56); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ IDNO:3); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ IDNO:7); a light chain CDR2 comprising RASNLEA (SEQ ID NO:8); and a lightchain CDR3 comprising QQSREYPYT (SEQ ID NO:9); wherein Xaa₁ is selectedfrom K, Q, H, and R; Xaa₂ is selected from Q, H, N, and R; and Xaa₃ isselected from G, E, T, S, A, and V. In certain embodiments, the antibodyis the huMov19 antibody, which is the above-described antibodycomprising the heavy chain CDR2 RIHPYDGDTFYNQKFQG (SEQ ID NO:2).

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huMov19 with up to four (i.e. 0, 1, 2, 3, or 4) conservativeamino acid substitutions per CDR. Thus, in certain embodiments theinvention provides humanized antibodies or antigen binding fragmentsthat specifically binds a human folate receptor 1, wherein the antibodycomprises: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID) NO:1), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ IDNO:2), or a variant thereof comprising 1, 2, 3, or 4 amino conservativeacid substitutions; and a heavy chain CDR3 comprising YDGSRAMDY (SEQ IDNO:3), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or (b) a light chain CDR1 comprisingKASQSVSFAGTSLMH (SEQ ID NO:7), or a variant thereof comprising 1, 2, 3,or 4 conservative amino acid substitutions; a light chain CDR2comprising RASNLEA (SEQ ID NO:8), or a variant thereof comprising 1, 2,3, or 4 conservative amino acid substitutions; and a light chain CDR3comprising QQSREYPYT (SEQ ID NO:9), or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions.

The invention also provides a humanized antibody (huFR1-21) or antigenbinding fragment thereof that specifically binds a human folate receptor1, wherein the antibody comprises: (a) a heavy chain CDR1 comprisingSSYGMS (SEQ ID NO:30); a heavy chain CDR2 comprising TISSGGSYTY (SEQ IDNO:31); and a heavy chain CDR3 comprising DGEGGLYAMDY (SEQ ID NO:32);and/or (b) a light chain CDR1 comprising KASDHINNWLA (SEQ ID NO:27); alight chain CDR2 comprising GATSLET (SEQ ID NO:28); and a light chainCDR3 comprising QQYWSTPFT (SEQ ID NO:29).

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huFR1-21 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. Thus, in certainembodiments the invention provides humanized antibodies or antigenbinding fragments that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSYGMS(SEQ ID NO:30) or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a heavy chain CDR2comprising TISSGGSYTY (SEQ ID NO:31) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and/or and a heavychain CDR3 comprising DGEGGLYAMDY (SEQ ID NO:32) or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or(b) a light chain CDR1 comprising KASDHINNWLA (SEQ ID NO:27) or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR2 comprising GATSLET (SEQ IDNO:28) or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a light chain CDR3 comprising QQYWSTPFT (SEQID NO:29) or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions.

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huFR1-48 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. Thus, in certainembodiments the invention provides humanized antibodies or antigenbinding fragments that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMQ(SEQ ID NO:60) or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a heavy chain CDR2comprising IYPGNGDSR (SEQ ID NO:61) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and/or and a heavychain CDR3 comprising RDGNYAAY (SEQ ID NO:62) or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or(b) a light chain CDR1 comprising RASENIYSNLA (SEQ ID NO:57) or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR2 comprising AATNLAD (SEQ IDNO:58) or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a light chain CDR3 comprising QHFWASPYT (SEQID NO:59) or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions.

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huFR1-49 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. Thus, in certainembodiments the invention provides humanized antibodies or antigenbinding fragments that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMY(SEQ ID NO:66) or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a heavy chain CDR2comprising AIYPGNSDTT (SEQ ID NO:67) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and/or and a heavychain CDR3 comprising RHDYGAMDY (SEQ ID NO:68) or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or(b) a light chain CDR1 comprising RASENIYTNLA (SEQ ID NO:63) or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR2 comprising TASNLAD (SEQ IDNO:64) or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a light chain CDR3 comprising QHFWVSPYT (SEQID NO:65) or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions.

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huFR1-57 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. Thus, in certainembodiments the invention provides humanized antibodies or antigenbinding fragments that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSFGMH(SEQ ID NO:72) or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a heavy chain CDR2comprising YISSGSSTIS (SEQ ID NO:73) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and/or and a heavychain CDR3 comprising EAYGSSMEY (SEQ ID NO:74) or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or(b) a light chain CDR1 comprising RASQNINNNLH (SEQ ID NO:69) or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR2 comprising YVSQSVS (SEQ IDNO:70) or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a light chain CDR3 comprising QQSNSWPHYT (SEQID NO:71) or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions.

In certain embodiments, the invention provides humanized antibodies orantigen binding fragments that specifically bind to FOLR1 that comprisethe CDRs of huFR1-65 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. Thus, in certainembodiments the invention provides humanized antibodies or antigenbinding fragments that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising TSYTMH(SEQ ID NO:78) or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and/or a heavy chain CDR2comprising YINPISGYTN (SEQ ID NO:79) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and/or and a heavychain CDR3 comprising GGAYGRKPMDY (SEQ ID NO:80) or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and/or(b) a light chain CDR1 comprising KASQNVGPNVA (SEQ ID NO:75) or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or a light chain CDR2 comprising SASYRYS (SEQ IDNO:76) or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or a light chain CDR3 comprising QQYNSYPYT (SEQID NO:77) or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions.

Polypeptides comprising one of the individual light chains or heavychains described herein, as well as polypeptides (e.g., antibodies)comprising both a light chain and a heavy chain are also provided. Thepolypeptides of SEQ ID NOs: 4 and 6 comprise the variable domain of theheavy chain of huMov19, and the heavy chain of huMov19, respectively.The polypeptides of SEQ ID NOs:10-13 comprise the variable domain lightchain version 1.00, the variable domain light chain version 1.60, thelight chain version 1.00, and the light chain version 1.60 of huMov19,respectively. The polypeptides of SEQ ID NOs: 42 and 46 comprise thevariable domain of the heavy chain of huFR1-21, and the heavy chain ofhuFR1-21, respectively. The polypeptides of SEQ ID NOs:41 and 45comprise the variable domain light chain and light chain of huFR1-21,respectively. The polypeptides of SEQ ID NOs: 97 and 113 comprise thevariable domain of the heavy chain of huFR1-48, and the heavy chain ofhuFR1-48, respectively. The polypeptides of SEQ ID NOs:96 and 112comprise the variable domain light chain and light chain of huFR1-48,respectively. The polypeptides of SEQ ID NOs: 99 and 115 comprise thevariable domain of the heavy chain of huFR1-49, and the heavy chain ofhuFR1-49, respectively. The polypeptides of SEQ ID NOs:98 and 114comprise the variable domain light chain and light chain of huFR1-49,respectively. The polypeptides of SEQ ID NOs: 101 and 117 comprise thevariable domain of the heavy chain of huFR1-57, and the heavy chain ofhuFR1-57, respectively. The polypeptides of SEQ ID NOs:100 and 116comprise the variable domain light chain and light chain of huFR1-57,respectively. The polypeptides of SEQ ID NOs:103 and 119 comprise thevariable domain of the heavy chain of huFR1-65, and the heavy chain ofhuFR1-65, respectively. The polypeptides of SEQ ID NOs:102 and 118comprise the variable domain light chain and light chain of huFR1-65,respectively.

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NO:4 or 6; and/or (b) apolypeptide having at least about 90% sequence identity to SEQ IDNOs:10-13. Also provided are polypeptides that comprise: (a) apolypeptide having about 90% sequence identity to SEQ ID NO: 42 or 46;and/or (b) a polypeptide having at least about 90% sequence identity toSEQ ID NOs: 41 and 45. Also provided are polypeptides that comprise: (a)a polypeptide having at least about 90% sequence identity to SEQ IDNO:97 or 113; and/or (b) a polypeptide having at least about 90%sequence identity to SEQ ID NOs:96 or 112. Also provided arepolypeptides that comprise: (a) a polypeptide having at least about 90%sequence identity to SEQ ID NO:99 or 115; and/or (b) a polypeptidehaving at least about 90% sequence identity to SEQ ID NOs:98 or 114.Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NO:101 or 117; and/or (b)a polypeptide having at least about 90% sequence identity to SEQ IDNOs:100 or 116. Also provided are polypeptides that comprise: (a) apolypeptide having at least about 90% sequence identity to SEQ ID NO:103or 119; and/or (b) a polypeptide having at least about 90% sequenceidentity to SEQ ID NOs:102 or 118. In certain embodiments, thepolypeptide comprises a polypeptide having at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NOs:4, 6, 10-13, 41, 42, 45 or 46. Thus, incertain embodiments, the polypeptide comprises (a) a polypeptide havingat least about 95% sequence identity to SEQ ID NO:4 or 6, and/or (b) apolypeptide having at least about 95% sequence identity to SEQ IDNOs:10-13. In certain embodiments, the polypeptide comprises (a) apolypeptide having at least about 95% sequence identity to SEQ ID NO:42or 46, and/or (b) a polypeptide having at least about 95% sequenceidentity to SEQ ID NOs:41 or 45. Also provided are polypeptides thatcomprise: (a) a polypeptide having at least about 95% sequence identityto SEQ ID NO:97 or 113; and/or (b) a polypeptide having at least about95% sequence identity to SEQ ID NOs:96 or 112. Also provided arepolypeptides that comprise: (a) a polypeptide having at least about 95%sequence identity to SEQ ID NO:99 or 115; and/or (b) a polypeptidehaving at least about 95% sequence identity to SEQ ID NOs:98 or 114.Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 95% sequence identity to SEQ ID NO:101 or 117; and/or (b)a polypeptide having at least about 95% sequence identity to SEQ ID NOs:100 or 116. Also provided are polypeptides that comprise: (a) apolypeptide having at least about 95% sequence identity to SEQ ID NO:103 or 119; and/or (b) a polypeptide having at least about 95% sequenceidentity to SEQ ID NOs: 102 or 118. In certain embodiments, thepolypeptide comprises (a) a polypeptide having the amino acid sequenceof SEQ ID NO: 4; and/or (b) a polypeptide having the amino acid sequenceof SEQ ID NO:10 or SEQ ID NO: 1. In certain embodiments, the polypeptidecomprises (a) a polypeptide having the amino acid sequence of SEQ IDNO:45; and/or (b) a polypeptide having the amino acid sequence of SEQ IDNO:46. In certain embodiments, the polypeptide comprises (a) apolypeptide having the amino acid sequence of SEQ ID NO: 6; and/or (b) apolypeptide having the amino acid sequence of SEQ ID NO:12 or SEQ IDNO:13. In certain embodiments, the polypeptide is an antibody and/or thepolypeptide specifically binds human folate receptor 1. In certainembodiments, the polypeptide is a humanized antibody that specificallybinds human folate receptor 1. For example, the invention provides anantibody or humanized antibody that specifically binds a human FOLR1that comprises (a) a polypeptide having the amino acid sequence of SEQID NO: 4; and (b) a polypeptide having the amino acid sequence of SEQ IDNO: 10 or SEQ ID NO: 11. In certain embodiments the polypeptidecomprising SEQ ID NO:4 is a heavy chain variable region. In certainembodiments, the polypeptide comprising SEQ ID NO:10 or 11 is a lightchain variable region. The invention also provides an antibody orhumanized antibody that specifically binds a human FOLR1 that comprises(a) a polypeptide having the amino acid sequence of SEQ ID NO: 6; and(b) a polypeptide having the amino acid sequence of SEQ ID NO:12 or SEQID NO:13. The invention also provides and antibody or humanized antibodythat specifically binds a human FOLR1 that comprises (a) a polypeptidehaving the amino acid sequence of SEQ ID NO:45; and (b) a polypeptidehaving the amino acid sequence of SEQ ID NO:46. The invention alsoprovides and antibody or humanized antibody that specifically binds ahuman FOLR1 that comprises (a) a polypeptide having the amino acidsequence of SEQ ID NO:112; and (b) a polypeptide having the amino acidsequence of SEQ ID NO:113. The invention also provides and antibody orhumanized antibody that specifically binds a human FOLR1 that comprises(a) a polypeptide having the amino acid sequence of SEQ ID NO:114; and(b) a polypeptide having the amino acid sequence of SEQ ID NO:115. Theinvention also provides and antibody or humanized antibody thatspecifically binds a human FOLR1 that comprises (a) a polypeptide havingthe amino acid sequence of SEQ ID NO:116; and (b) a polypeptide havingthe amino acid sequence of SEQ ID NO:117. The invention also providesand antibody or humanized antibody that specifically binds a human FOLR1that comprises (a) a polypeptide having the amino acid sequence of SEQID NO: 118; and (b) a polypeptide having the amino acid sequence of SEQID NO:119. In certain embodiments, the polypeptide having a certainpercentage of sequence identity to SEQ ID NOs: 4, 6, 10-13, 41, 42, 45,46, 96-103 and 112-119 differs from SEQ ID NO: 4, 6, 10-13, 41, 42, 45,46, 96-103 and 112-119 by conservative amino acid substitutions only.

In certain embodiments, the FOLR1-binding agent comprises, consistsessentially of, or consists of an anti-FOLR1 antibody selected from thegroup consisting of huMov19, FR-1-21, FR1-48, FR1-49, FR1-57, and FR1-65antibodies.

In certain embodiments, the huMov19 antibody is encoded by the plasmidsdeposited with the American Type Culture Collection (ATCC) on Apr. 7,2010 and having ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.

In certain embodiments, the FR-1-21 antibody is encoded by the plasmidsdeposited with the ATCC on Apr. 7, 2010, and assigned depositdesignation numbers PTA-10775 and 10776.

In certain embodiments, the humanized antibodies bind FOLR1 withsubstantially the same affinity as the antibody chimeric Mov19. Theaffinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method well known in the art, e.g.flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), orradioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Directbinding assays as well as competitive binding assay formats can bereadily employed. (See, for example, Berzofsky, et al.,“Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H.Freeman and Company: New York, N.Y. (1992); and methods describedherein. The measured affinity of a particular antibody-antigeninteraction can vary if measured under different conditions (e.g., saltconcentration, pH, temperature). Thus, measurements of affinity andother antigen-binding parameters (e.g., KD or Kd, K_(on), K_(off)) aremade with standardized solutions of antibody and antigen, and astandardized buffer, as known in the art and such as the bufferdescribed herein.

In one aspect, binding assays can be performed using flow cytometry oncells expressing the FOLR1 antigen on the surface. For example,FOLR1-positive cells such as SKOV3 were incubated with varyingconcentrations of anti-FOLR1 antibodies using 1×105 cells per sample in100 μL FACS buffer (RPMI-1640 medium supplemented with 2% normal goatserum). Then, the cells were pelleted, washed, and incubated for 1 hwith 100 μL of FITC-conjugated goat-anti-mouse or goat-anti-humanIgG-antibody (such as is obtainable from, for example JacksonLaboratory, 6 μg/mL in FACS buffer). The cells were pelleted again,washed with FACS buffer and resuspended in 200 μL of PBS containing 1%formaldehyde. Samples were acquired, for example, using a FACSCaliburflow cytometer with the HTS multiwell sampler and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US). For each samplethe mean fluorescence intensity for FL1 (MFI) was exported and plottedagainst the antibody concentration in a semi-log plot to generate abinding curve. A sigmoidal dose-response curve is fitted for bindingcurves and EC50 values are calculated using programs such as GraphPadPrism v4 with default parameters (GraphPad software, San Diego, Calif.).EC50 values can be used as a measure for the apparent dissociationconstant “Kd” or “KD” for each antibody.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human FOLR1 isa humanized antibody. In certain embodiments, such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject.

Methods for engineering, humanizing or resurfacing non-human or humanantibodies can also be used and are well known in the art. A humanized,resurfaced or similarly engineered antibody can have one or more aminoacid residues from a source that is non-human, e.g., but not limited to,mouse, rat, rabbit, non-human primate or other mammal. These non-humanamino acid residues are replaced by residues that are often referred toas “import” residues, which are typically taken from an “import”variable, constant or other domain of a known human sequence.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing FOLR1 binding. Accordingly, partor all of the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions can be replacedwith human or other amino acids.

Antibodies can also optionally be humanized, resurfaced, engineered orhuman antibodies engineered with retention of high affinity for theantigen FOLR1 and other favorable biological properties. To achieve thisgoal, humanized (or human) or engineered anti-FOLR1 antibodies andresurfaced antibodies can be optionally prepared by a process ofanalysis of the parental sequences and various conceptual humanized andengineered products using three-dimensional models of the parental,engineered, and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen, such as FOLR1. In this way, framework (FR) residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Humanization, resurfacing or engineering of antibodies of the presentinvention can be performed using any known method, such as but notlimited to those described in, Winter (Jones et al., Nature 321:522(1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al.,Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993);Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc.Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862;5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;5,225,539; 4,816,567; PCT/: US98/16280; US96/18978; US91/09630;US91/05939; US94/01234; GB89/01334; GB91/01134; GB92/01755; WO90/14443;WO90/14424; WO90/14430; EP 229246; U.S. Pat. Nos. 7,557,189; 7,538,195;and 7,342,110, each of which is entirely incorporated herein byreference, including the references cited therein.

In certain alternative embodiments, the antibody to FOLR1 is a humanantibody. Human antibodies can be directly prepared using varioustechniques known in the art. Immortalized human B lymphocytes immunizedin vitro or isolated from an immunized individual that produce anantibody directed against a target antigen can be generated (See, e.g.,Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S.Pat. No. 5,750,373). Also, the human antibody can be selected from aphage library, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., 1992,Bio/Technology 10:779-783, incorporated by reference in its entirety)are known in the art and can be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a human folate receptor 1. Bispecific antibodies areantibodies that are capable of specifically recognizing and binding atleast two different epitopes. The different epitopes can either bewithin the same molecule (e.g. the same human folate receptor 1) or ondifferent molecules such that both, for example, the antibodies canspecifically recognize and bind a human folate receptor 1 as well as,for example, 1) an effector molecule on a leukocyte such as a T-cellreceptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) acytotoxic agent as described in detail below.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodimentsthe antibodies to FOLR1 are multispecific.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to human folate receptor1 (see U.S. Pat. No. 4,946,778). In addition, methods can be adapted forthe construction of Fab expression libraries (Huse, et al., Science246:1275-1281 (1989)) to allow rapid and effective identification ofmonoclonal Fab fragments with the desired specificity for a folate 1receptor, or derivatives, fragments, analogs or homologs thereof.Antibody fragments can be produced by techniques in the art including,but not limited to: (a) a F(ab′)2 fragment produced by pepsin digestionof an antibody molecule; (b) a Fab fragment generated by reducing thedisulfide bridges of an F(ab′)2 fragment, (c) a Fab fragment generatedby the treatment of the antibody molecule with papain and a reducingagent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman FOLR1. In this regard, the variable region can comprise or bederived from any type of mammal that can be induced to mount a humoralresponse and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and in certain embodiments from an antibodyfrom a different species. It may not be necessary to replace all of theCDRs with the complete CDRs from the donor variable region to transferthe antigen binding capacity of one variable domain to another. Rather,it may only be necessary to transfer those residues that are necessaryto maintain the activity of the antigen binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein can comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2 or CH3) and/or to the lightchain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g. 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the FOLR1-binding antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain canreduce Fc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases it may be that constantregion modifications, consistent with this invention, moderatecomplement binding and thus reduce the serum half life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to eliminate disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. Similarly,modifications to the constant region in accordance with this inventioncan easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain embodiments, a FOLR1-binding agent that is an antibody doesnot have one or more effector functions. For instance, in someembodiments, the antibody has no antibody-dependent cellular cytoxicity(ADCC) activity and/or no complement-dependent cytoxicity (CDC)activity. In certain embodiments, the antibody does not bind to an Fcreceptor and/or complement factors. In certain embodiments, the antibodyhas no effector function.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it may bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement C1Q binding) to bemodulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it can be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human FOLR1. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against a human folate receptorprotein. Such mutants include deletions, insertions, inversions,repeats, and type substitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human FOLR1. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-FOLR1 antibody, or fragment thereof,operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it can include an N-terminalmethionine residue. This residue can optionally be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovims andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a FOLR1-binding polypeptide orantibody (or a FOLR1 protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N. Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines includeHEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, describedby Gluzman (Cell 23:175, 1981), and other cell lines including, forexample, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHKcell lines. Mammalian expression vectors can comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a FOLR1-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNo. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the FOLR1-binding agent is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gillet al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety. In certainembodiments, phage display technology has been used to identify/producethe FOLR1-binding polypeptide. In certain embodiments, the polypeptidecomprises a protein scaffold of a type selected from the groupconsisting of protein A, a lipocalin, a fribronectin domain, an ankyrinconsensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinFOLR1-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., J. Comb. Chem, 10:345-354 (2008), Dolle et al, J. Comb.Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8:1383-404(2001), each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. No. 5,270,163, U.S. Pat.No. 5,683,867, U.S. Pat. No. 5,763,595, U.S. Pat. No. 6,344,321, U.S.Pat. No. 7,368,236, U.S. Pat. No. 5,582,981, U.S. Pat. No. 5,756,291,U.S. Pat. No. 5,840,867, U.S. Pat. No. 7,312,325, U.S. Pat. No.7,329,742, International Patent Publication No. WO 02/077262,International Patent Publication No. WO 03/070984, U.S. PatentApplication Publication No. 2005/0239134, U.S. Patent ApplicationPublication No. 2005/0124565, and U.S. Patent Application PublicationNo. 2008/0227735, each of which is incorporated by reference herein inits entirety.

III. IMMUNOCONJUGATES

The present invention is also directed to conjugates (also referred toherein as immunoconjugates), comprising the anti-FOLR1 antibodies,antibody fragments, functional equivalents, improved antibodies andtheir aspects as disclosed herein, linked or conjugated to a cytotoxin(drug) or prodrug. Thus, in a certain embodiment, the invention providesan immunoconjugate comprising a humanized antibody or antigen bindingfragment thereof that specifically binds a human folate receptor 1,wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN(SEQ ID NO:1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa₁FXaa₂Xaa₃(SEQ ID NO:56); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ IDNO:3); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ IDNO:7); a light chain CDR2 comprising RASNLEA (SEQ ID NO:8); and a lightchain CDR3 comprising QQSREYPYT (SEQ ID NO:9); wherein Xaa₁ is selectedfrom K, Q, H, and R; Xaa₂ is selected from Q, H, N, and R; and Xaa₃ isselected from G, E, T, S, A, and V. In certain embodiments, the antibodyis the huMov19 antibody, which is the above-described antibodycomprising the heavy chain CDR2 RIHPYDGDTFYNQKFQG (SEQ ID NO:2). Inother embodiments, the antibody is FR1-21 and comprises (a) a heavychain CDR1 comprising SSYGMS (SEQ ID NO:30); a heavy chain CDR2comprising TISSGGSYTY (SEQ ID NO:31); and/or a heavy chain CDR3comprising DGEGGLYAMDY (SEQ ID NO:32); and (b) a light chain CDR1comprising KASDHINNWLA (SEQ ID NO:27); a light chain CDR2 comprisingGATSLET (SEQ ID NO:28); and a light chain CDR3 comprising QQYWSTPFT (SEQID NO:29). In other embodiments, the antibody is FR1-48 and comprises:(a) a heavy chain CDR1 comprising TNYWMQ (SEQ ID NO:60); a heavy chainCDR2 comprising AIYPGNGDSR (SEQ ID NO:61); and/or a heavy chain CDR3comprising RDGNYAAY (SEQ ID NO:62); and/or (b) a light chain CDR1comprising RASENIYSNLA (SEQ ID NO:57); a light chain CDR2 comprisingAATNLAD (SEQ ID NO:58); and a light chain CDR3 comprising QHFWASPYT (SEQID NO:59). In other embodiments, the antibody is FR1-49 and comprises:(a) a heavy chain CDR1 comprising TNYWMY (SEQ ID NO:66); a heavy chainCDR2 comprising AIYPGNSDTT (SEQ ID NO:67); and/or a heavy chain CDR3comprising RHDYGAMDY (SEQ ID NO:68); and/or (b) a light chain CDR1comprising RASENIYTNLA (SEQ ID NO:63); a light chain CDR2 comprisingTASNLAD (SEQ ID NO:64); and a light chain CDR3 comprising QHFWVSPYT (SEQID NO:65). In other embodiments, the antibody is FR1-57 and comprises:(a) a heavy chain CDR1 comprising SSFGMH (SEQ ID NO:72); a heavy chainCDR2 comprising YISSGSSTIS (SEQ ID NO:73); and/or a heavy chain CDR3comprising EAYGSSMEY (SEQ ID NO:74); and/or (b) a light chain CDR1comprising RASQNINNNLH (SEQ ID NO:69); a light chain CDR2 comprisingYVSQSVS (SEQ ID NO:70); and a light chain CDR3 comprising QQSNSWPHYT(SEQ ID NO:71). In yet another embodiment, the antibody is FR1-65 andcomprises: (a) a heavy chain CDR1 comprising TSYTMH (SEQ ID NO:78); aheavy chain CDR2 comprising YINPISGYTN (SEQ ID NO:79); and/or a heavychain CDR3 comprising GGAYGRKPMDY (SEQ ID NO:80); and/or (b) a lightchain CDR1 comprising KASQNVGPNVA (SEQ ID NO:75); a light chain CDR2comprising SASYRYS (SEQ ID NO:76); and a light chain CDR3 comprisingQQYNSYPYT (SEQ ID NO:77).

Suitable drugs or prodrugs are known in the art. In certain embodiments,drugs or prodrugs are cytotoxic agents. The cytotoxic agent used in thecytotoxic conjugate of the present invention can be any compound thatresults in the death of a cell, or induces cell death, or in some mannerdecreases cell viability, and includes, for example, maytansinoids andmaytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065analogs, duocarmycins and duocarmycin analogs, enediynes, such ascalicheamicins, dolastatin and dolastatin analogs including auristatins,tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin,carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine,melphalan, mitomycin C, chlorambucil and morpholino doxorubicin. Incertain embodiments, the cytotoxic agents are maytansinoids andmaytansinoids analogs.

Such conjugates can be prepared by using a linking group in order tolink a drug or prodrug to the antibody or functional equivalent.Suitable linking groups are well known in the art and include, forexample, disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.

The drug or prodrug can, for example, be linked to the anti-FOLR1antibody or fragment thereof through a disulfide bond. The linkermolecule or crosslinking agent comprises a reactive chemical group thatcan react with the anti-FOLR1 antibody or fragment thereof. In certainembodiments, reactive chemical groups for reaction with the cell-bindingagent are N-succinimidyl esters and N-sulfosuccinimidyl esters.Additionally the linker molecule comprises a reactive chemical group, incertain embodiments a dithiopyridyl group that can react with the drugto form a disulfide bond. In certain embodiments, linker moleculesinclude, for example, N-succinimidyl 3-(2-pyridyldithio) propionate(SPDP) (see, e.g., Carlsson et al., Biochem. J., 173: 723-737 (1978)),N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat.No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate(sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example,the antibody or cell binding agent can be modified with crosslinkingreagents and the antibody or cell binding agent containing free orprotected thiol groups thus derived is then reacted with a disulfide- orthiol-containing maytansinoid to produce conjugates. The conjugates canbe purified by chromatography, including but not limited to HPLC,size-exclusion, adsorption, ion exchange and affinity capture, dialysisor tangential flow filtration. In certain embodiments, the anti-FOLR1antibody is linked to the cytoxin via a SPDB or sulfo-SPDB linker. In acertain embodiment, the huMov19 antibody is linked to a cytotoxin via aSPDB or sulfo-SPDB linker.

In another aspect of the present invention, the anti-FOLR1 antibody islinked to cytotoxic drugs via disulfide bonds and a polyethylene glycolspacer in enhancing the potency, solubility or the efficacy of theimmunoconjugate. Such cleavable hydrophilic linkers are described inWO2009/0134976. The additional benefit of this linker design is thedesired high monomer ratio and the minimal aggregation of theantibody-drug conjugate. Specifically contemplated in this aspect areconjugates of cell-binding agents and drugs linked via disulfide group(—S—S—) bearing polyethylene glycol spacers ((CH₂CH₂O)_(n=1-14)) with anarrow range of drug load of 2-8 are described that show relatively highpotent biological activity toward cancer cells and have the desiredbiochemical properties of high conjugation yield and high monomer ratiowith minimal protein aggregation.

Specifically contemplated in this aspect is an anti-FOLR1 antibody drugconjugate of formula (I) or a conjugate of formula (I′):

A-[X_(l)—(—CH₂—CH₂O—)_(n)—Y—C]_(m)  (I)

[C—Y—(—CH₂—CH₂O—)_(n)—X_(l)]_(m)-A  (I′)

-   -   wherein:    -   A represents an anti-FOLR1 antibody or fragment;    -   C represents a cytotoxin or drug;    -   X represents an aliphatic, an aromatic or a heterocyclic unit        attached to the cell-binding agent via a thioether bond, an        amide bond, a carbamate bond, or an ether bond;    -   Y represents an aliphatic, an aromatic or a heterocyclic unit        attached to the drug via a disulfide bond;    -   l is 0 or 1;    -   m is an integer from 2 to 8; and    -   n is an integer from 1 to 24.    -   In certain embodiments, m is an integer from 2 to 6.    -   In certain embodiments, m is an integer from 3 to 5.

Also, In certain embodiments, n is an integer form 2 to 8.Alternatively, as disclosed in, for example, U.S. Pat. Nos. 6,441,163and 7,368,565, the drug can be first modified to introduce a reactiveester suitable to react with a cell-binding agent. Reaction of thesedrugs containing an activated linker moiety with a cell-binding agentprovides another method of producing a cell-binding agent drugconjugate. Maytansinoids can also be linked to anti-FOLR1 antibody orfragment using PEG linking groups, as set forth for example in U.S. Pat.No. 6,716,821. These PEG non-cleavable linking groups are soluble bothin water and in non-aqueous solvents, and can be used to join one ormore cytotoxic agents to a cell binding agent. Exemplary PEG linkinggroups include heterobifunctional PEG linkers that react with cytotoxicagents and cell binding agents at opposite ends of the linkers through afunctional sulfhydryl or disulfide group at one end, and an active esterat the other end. As a general example of the synthesis of a cytotoxicconjugate using a PEG linking group, reference is again made to U.S.Pat. No. 6,716,821 which is incorporated entirely by reference herein.Synthesis begins with the reaction of one or more cytotoxic agentsbearing a reactive PEG moiety with a cell-binding agent, resulting indisplacement of the terminal active ester of each reactive PEG moiety byan amino acid residue of the cell binding agent, to yield a cytotoxicconjugate comprising one or more cytotoxic agents covalently bonded to acell binding agent through a PEG linking group. Alternatively, the cellbinding can be modified with the bifunctional PEG crosslinker tointroduce a reactive disulfide moiety (such as a pyridyldisulfide),which can then be treated with a thiol-containing maytansinoid toprovide a conjugate. In another method, the cell binding can be modifiedwith the bifunctional PEG crosslinker to introduce a thiol moiety whichcan then can be treated with a reactive disulfide-containingmaytansinoid (such as a pyridyldisulfide), to provide a conjugate.

Antibody-maytansinoid conjugates with non-cleavable links can also beprepared. Such crosslinkers are described in the art (seeThermoScientific Pierce Crosslinking Technical Handbook and US PatentApplication Publication No. 2005/0169933) and include but are notlimited to, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate(SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoicacid N-succinimidyl ester (KMUA), β-maleimidopropanoic acidN-succinimidyl ester (BMPS), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester (AMAS),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI),N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl3-(bromoacetamido)propionate (SBAP). In certain embodiments, theantibody is modified with crosslinking reagents such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS orsuccinimidyl-iodoacetate, as described in the literature, to introduce1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101:395-399(1979); Hashida et al, J. Applied Biochem., 56-63 (1984); and Liu et al,Biochem., 18:690-697 (1979)). The modified antibody is then reacted withthe thiol-containing maytansinoid derivative to produce a conjugate. Theconjugate can be purified by gel filtration through a Sephadex G25column or by dialysis or tangential flow filtration. The modifiedantibodies are treated with the thiol-containing maytansinoid (1 to 2molar equivalent/maleimido group) and antibody-maytansinoid conjugatesare purified by gel filtration through a Sephadex G-25 column,chromatography on a ceramic hydroxyapatite column, dialysis ortangential flow filtration or a combination of methods thereof.Typically, an average of 1-10 maytansinoids per antibody are linked. Onemethod is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again conjugates with 1 to 10 drug molecules per antibody moleculeresult. Maytansinoid conjugates of antibodies, antibody fragments,protein hormones, protein growth factors and other proteins are made inthe same way.

In another aspect of the invention, the FOLR1 antibody (e.g. huMov19,FR1-21, FR1-48, FR1-49, FR1-57, or FR1-65) is linked to the drug via anon-cleavable bond through the intermediacy of a PEG spacer. Suitablecrosslinking reagents comprising hydrophilic PEG chains that formlinkers between a drug and the anti-FOLR1 antibody or fragment are alsowell known in the art, or are commercially available (for example fromQuanta Biodesign, Powell, Ohio). Suitable PEG-containing crosslinkerscan also be synthesized from commercially available PEGs themselvesusing standard synthetic chemistry techniques known to one skilled inthe art. The drugs can be reacted with bifunctional PEG-containing crosslinkers to give compounds of the following formula,Z—X_(l)—(—CH₂—CH₂—O—)_(n)—Y_(p)-D, by methods described in detail in USPatent Publication 20090274713 and in WO2009/0134976, which can thenreact with the cell binding agent to provide a conjugate. Alternatively,the cell binding can be modified with the bifunctional PEG crosslinkerto introduce a thiol-reactive group (such as a maleimide orhaloacetamide) which can then be treated with a thiol-containingmaytansinoid to provide a conjugate. In another method, the cell bindingcan be modified with the bifunctional PEG crosslinker to introduce athiol moiety which can then be treated with a thiol-reactivemaytansinoid (such as a maytansinoid bearing a maleimide orhaloacetamide), to provide a conjugate.

Accordingly, another aspect of the present invention is an anti-FOLR1antibody drug conjugate of formula (II) or of formula (II′):

A-[X_(l)—(—CH₂—CH₂—O—)_(n)—Y_(p)—C]_(m)  (II)

[C—Y_(p)—(—CH₂—CH₂—O—)_(n)—X_(l)]_(m)-A  (II′)

-   -   wherein, A represents an anti-FOLR1 antibody or fragment;    -   C represents a cytotoxin or drug;    -   X represents an aliphatic, an aromatic or a heterocyclic unit        bonded to the cell-binding agent via a thioether bond, an amide        bond, a carbamate bond, or an ether bond;    -   Y represents an aliphatic, an aromatic, or a heterocyclic unit        bonded to the drug via a covalent bond selected from the group        consisting of a thioether bond, an amide bond, a carbamate bond,        an ether bond, an amine bond, a carbon-carbon bond and a        hydrazone bond;    -   l is 0 or 1;    -   p is 0 or 1;    -   m is an integer from 2 to 15; and    -   n is an integer from 1 to 2000.    -   In a certain embodiment, m is an integer from 2 to 8; and    -   n is an integer from 1 to 24.    -   In a certain embodiment, m is an integer from 2 to 6.    -   In a certain embodiment, n is an integer from 2 to 8.

In a certain embodiment, m is an integer from 3 to 5. In a certainembodiment, the antibody is huMov19. In another embodiment, the antibodyis FR-1-21. In another embodiment, the antibody is FR-1-48. In anotherembodiment, the antibody is FR-1-49. In another embodiment, the antibodyis FR-1-57. In another embodiment, the antibody is FR-1-65.

Examples of suitable PEG-containing linkers include linkers having anN-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reactionwith the anti-FOLR1 antibody or fragment thereof, as well as amaleimido- or haloacetyl-based moiety for reaction with the compound. APEG spacer can be incorporated into any crosslinker known in the art bythe methods described herein.

Many of the linkers disclosed herein are described in detail in U.S.Patent Publication Nos. 20050169933 and 20090274713, and inWO2009/0134976; the contents of which are entirely incorporated hereinby reference.

The present invention includes aspects wherein about 2 to about 8 drugmolecules (“drug load”), for example, maytansinoid, are linked to ananti-FOLR1 antibody or fragment thereof, the anti-tumor effect of theconjugate is much more efficacious as compared to a drug load of alesser or higher number of drugs linked to the same cell binding agent.“Drug load”, as used herein, refers to the number of drug molecules(e.g., a maytansinoid) that can be attached to a cell binding agent(e.g., an anti-FOLR1 antibody or fragment thereof). In one aspect thenumber of drug molecules that can be attached to a cell binding agentcan average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1). In certain embodiments, the drug isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl) maytansine(DM4). Thus, in a certain embodiment, the antibody huMov19 is conjugatedto DM1 or DM4. In another embodiment, the antibody FR-1-21 is conjugatedto DM1 or DM4. In another embodiment, the antibody FR-1-48 is conjugatedto DM1 or DM4. In another embodiment, the antibody FR-1-49 is conjugatedto DM1 or DM4. In another embodiment, the antibody FR-1-57 is conjugatedto DM1 or DM4. In another embodiment, the antibody FR-1-65 is conjugatedto DM1 or DM4.

Thus, in one aspect, an immunoconjugate comprises 1 maytansinoid perantibody. In another aspect, an immunoconjugate comprises 2maytansinoids per antibody. In another aspect, an immunoconjugatecomprises 3 maytansinoids per antibody. In another aspect, animmunoconjugate comprises 4 maytansinoids per antibody. In anotheraspect, an immunoconjugate comprises 5 maytansinoids per antibody. Inanother aspect, an immunoconjugate comprises 6 maytansinoids perantibody. In another aspect, an immunoconjugate comprises 7maytansinoids per antibody. In another aspect, an immunoconjugatecomprises 8 maytansinoids per antibody.

In one aspect, an immunoconjugate comprises about 1 to about 8maytansinoids per antibody. In another aspect, an immunoconjugatecomprises about 2 to about 7 maytansinoids per antibody. In anotheraspect, an immunoconjugate comprises about 2 to about 6 maytansinoidsper antibody. In another aspect, an immunoconjugate comprises about 2 toabout 5 maytansinoids per antibody. In another aspect, animmunoconjugate comprises about 3 to about 5 maytansinoids per antibody.In another aspect, an immunoconjugate comprises about 3 to about 4maytansinoids per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drugmolecules (e.g., maytansinoids) attached per antibody. In one aspect, acomposition comprising immunoconjugates has an average of about 1 toabout 8 drug molecules (e.g., maytansinoids) per antibody. In oneaspect, a composition comprising immunoconjugates has an average ofabout 2 to about 7 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 6 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 4 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3.5 to about 4 drug molecules (e.g., maytansinoids) per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2±0.5, about 2.5±0.5, about 3±0.5, about 3.5±0.5, about 4±0.5,about 4.5±0.5, about 5±0.5, about 5.5±0.5, about 6±0.5, about 6.5±0.5,about 7±0.5, about 7.5±0.5, or about 8±0.5 drug molecules (e.g.,maytansinoids) attached per antibody. In one aspect, a compositioncomprising immunoconjugates has an average of about 3.5±0.5 drugmolecules (e.g., maytansinoids) per antibody.

The anti-FOLR1 antibody or fragment thereof can be modified by reactinga bifunctional crosslinking reagent with the anti-FOLR1 antibody orfragment thereof, thereby resulting in the covalent attachment of alinker molecule to the anti-FOLR1 antibody or fragment thereof. As usedherein, a “bifunctional crosslinking reagent” is any chemical moietythat covalently links a cell-binding agent to a drug, such as the drugsdescribed herein. In another method, a portion of the linking moiety isprovided by the drug. In this respect, the drug comprises a linkingmoiety that is part of a larger linker molecule that is used to join thecell-binding agent to the drug. For example, to form the maytansinoidDM1, the side chain at the C-3 hydroxyl group of maytansine is modifiedto have a free sulfhydryl group (SH). This thiolated form of maytansinecan react with a modified cell-binding agent to form a conjugate.Therefore, the final linker is assembled from two components, one ofwhich is provided by the crosslinking reagent, while the other isprovided by the side chain from DM1.

The drug molecules can also be linked to the antibody molecules throughan intermediary carrier molecule such as serum albumin.

As used herein, the expression “linked to a cell-binding agent” or“linked to an anti-FOLR1 antibody or fragment” refers to the conjugatemolecule comprising at least one drug derivative bound to a cell-bindingagent anti-FOLR1 antibody or fragment via a suitable linking group, or aprecursor thereof. In certain embodiments, the linking group is SMCC.

In certain embodiments, cytotoxic agents useful in the present inventionare maytansinoids and maytansinoid analogs. Examples of suitablemaytansinoids include esters of maytansinol and maytansinol analogs.Included are any drugs that inhibit microtubule formation and that arehighly toxic to mammalian cells, as are maytansinol and maytansinolanalogs.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219;4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598;4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410;7,276,497 and 7,473,796.

In a certain embodiment, the immunoconjugates of the invention utilizethe thiol-containing maytansinoid (DM1), formally termedN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, as thecytotoxic agent. DM1 is represented by the following structural formula(III):

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoidN^(2′)-deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine(e.g., DM4) as the cytotoxic agent. DM4 is represented by the followingstructural formula (IV):

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN^(2′)-deacetyl-N-²′(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the following structural formula (V):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of U.S. Pat. Nos. 5,208,020 and7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In certain embodiments, the C-3 position isutilized. In certain embodiments, the C-3 position of maytansinol isutilized.

Structural representations of certain conjugates are shown below:

In a certain embodiment, the antibody is huMov19. In another embodiment,the antibody is FR1-21.

Several descriptions for producing such antibody-maytansinoid conjugatesare provided in U.S. Pat. Nos. 6,333,410, 6,441,163, 6,716,821, and7,368,565, each of which is incorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer can be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate can then be purified by gel filtration.The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. An average of 1-10 maytansinoidmolecules/antibody molecule is used and an average of 2-5 is also usedin certain embodiments. The average number of maytansinoidmolecules/antibody can be, for example, about 1-10, 2-5, 3-4, 3.5-4 or3.5. In one aspect, the average number of maytansinoidmolecules/antibody is about 3.5±0.5. In one aspect, the average numberof maytansinoid molecules/antibody is about 3.5-4.

Conjugates of antibodies with maytansinoid drugs can be evaluated fortheir ability to suppress proliferation of various unwanted cell linesin vitro. For example, cell lines such as the human KB cell line, caneasily be used for the assessment of cytotoxicity of these compounds.Cells to be evaluated can be exposed to the compounds for 4 to 5 daysand the surviving fractions of cells measured in direct assays by knownmethods. IC₅₀ values can then be calculated from the results of theassays.

Benzodiazepine compounds described, for example, in U.S. PatentApplication Publication No. 2010/0203007 (e.g., indolinobenzodiazepinesor oxazolidinobenzodiazepines), derivatives thereof, intermediatesthereof, may also be used to prepare anti-FOLR1 antibody fragment orconjugates.

Useful benzodiazepines include compounds of formula (XIV), (XV) and(XVI), in which the dimer compounds optionally bear a linking group thatallows for linkage to cell binding agents.

wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting moiety that converts the compoundinto a prodrug;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxyl amine represented by NR′R″, amide represented by —NRCOR′,a peptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand are selected from H, substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to2000, aryl having from 6 to 10 carbon atoms, heterocyclic ring havingfrom 3 to 10 carbon atoms wherein the substituent is selected fromhalogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′,a sulfone represented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, asulfonamide represented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ orOCONR₁₁R₁₂, wherein the definitions of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ areas given above, optionally R″ is OH;

W is C═O, C═S, CH₂, BH, SO or SO₂;

R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently selectedfrom H, substituted or unsubstituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000, or asubstituent selected from a halogen, guanidinium [—NH(C═NH)NH₂], OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, aryl having from 6 to 10 carbon atoms,heterocyclic ring having from 3 to 10 carbon atoms, optionally R₁₀ isSR₁₃ or COR₁₃, wherein R₁₃ is selected from linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, aryl having from 6 to 10 carbon atoms, heterocyclic ring havingfrom 3 to 10 carbon atoms, optionally R₁₁ is OR₁₄, wherein R₁₄ has thesame definition as R, optionally, any one of R₁, R₂, R₃, R₄, R₁′, R₂′,R₃′, or R₄′ is a linking group that enables linkage to a cell bindingagent via a covalent bond or is selected from a polypyrrolo,poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl,poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing alinking group that enables linkage to a cell binding agent;Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;R₆ is OR, SR or NRR′, wherein R and R′ have the same definition as givenabove;X′ is selected from CH₂, NR, CO, BH, SO or SO₂ wherein R has the samedefinition as given above;Y′ is O, CH₂, NR or S, wherein R has the same definition as given above;Z′ is CH₂ or (CH₂)_(n), wherein n is 2, 3 or 4, provided that X′, Y′ andZ′ are not all CH₂ at the same time;A and A′ are the same or different and are selected from O, —CRR′O, S,—CRR′S, —NR₁₅ or CRR′NHR₁₅, wherein R and R′ have the same definition asgiven above and wherein R₁₅ has the same definition as given above forR;D and D′ are same or different and independently selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms, optionally substituted with any one of halogen, OR₇, NR₈R₉, NO₂,NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfone represented by—SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented bySO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁ or OCONR₁₁R₁₂, wherein thedefinitions of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as given above, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;L is an optional phenyl group or a heterocycle ring having from 3 to 10carbon atoms that is optionally substituted, wherein the substituent isa linking group that enables linkage to a cell binding agent via acovalent bond, or is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, optionallysubstituted with any one of halogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, asulfoxide represented by SOR′, a sulfone represented by —SO₂R′, asulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented by SO₂NRR′,cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂, wherein the definitionsof R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as given above, a polyethyleneglycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000;optionally, L itself is a linking group that enables linkage to a cellbinding agent via a covalent bond; or their pharmaceutically acceptablesolvates, salts, hydrates or hydrated salts, their optical isomers,racemates, diastereomers, enantiomers or the polymorphic crystallinestructures of these compounds; provided that the compound has no morethan one linking group that enables linkage to a cell binding agent viaa covalent bond.

In one aspect, the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting group that converts the compoundinto a prodrug;

Y is selected from —OR, NR′R″, a sulfite —SO₃, or a bisulfite —OSO₃,wherein R is selected from H, linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, aryl having from6 to 10 carbon atoms, heterocyclic ring having from 3 to 10 carbonatoms;

W is C═O, CH₂ or SO₂;

R₁, R₂, R₃, R₄, R₁′. R₂′. R₃′ and R₄′ are each independently selectedfrom H, NO₂ or a linking group that enables linkage to a cell bindingagent via a covalent bond;R₆ is OR₁₈, wherein R₁₈ has the same definition as R;Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;X′ is selected from CH₂, or C═O;Y′ is O, NR, or S, wherein R is defined as above;

Z′ is CH₂ or (CH₂)₂;

A and A′ are each O;D and D′ are same or different and independently selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms;L is an optional phenyl group or a heterocycle ring having from 3 to 10carbon atoms that is optionally substituted, wherein the substituent isa linking group that enables linkage to a cell binding agent via acovalent bond, or is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, optionallysubstituted with any one of halogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, asulfoxide represented by SOR′, a sulfone represented by —SO₂R′, asulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented by SO₂NRR′,cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂, a polyethylene glycolunit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000; optionally, Litself is a linking group that enables linkage to a cell binding agentvia a covalent bond; or their pharmaceutically acceptable solvates,salts, hydrates or hydrated salts, their optical isomers, racemates,diastereomers, enantiomers or the polymorphic crystalline structures ofthese compounds.

In another aspect the compound is represented by formula (XVII):

wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting group that converts the compoundinto a prodrug, and Y is selected from OH, an ether represented by —OR,a sulfite —SO₃, or a bisulfite —OSO₃, wherein R is selected from linear,branched or cyclic alkyl, alkenyl or alkynyl bearing from 1 to 10 carbonatomsone of R2, R3 is a linking group that enables linkage to a cell bindingagent via a covalent bond and the other is H,one of L′, L″ or L′″ is a linking group that enables linkage to a cellbinding agent, while the others are H; L′can be the linking group and Gis CH or N. Other examples are described in U.S. Patent Application No.61/150,201, the entire content of which is incorporated herein byreference. Thus, in a certain embodiment, the antibody huMov19 isconjugated to a benzodiazepene having a structure shown in XIX-XXIIabove. In another embodiment, the antibody FR-1-21 is conjugated to abenzodiazepene having a structure shown in XIX-XXII above.

IV. POLYNUCLEOTIDES

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds a human FOLR1 receptor or a fragment of such a polypeptide. Forexample, the invention provides a polynucleotide comprising a nucleicacid sequence that encodes an antibody to a human FOLR1 or encodes afragment of such an antibody. The polynucleotides of the invention canbe in the form of RNA or in the form of DNA. DNA includes cDNA, genomicDNA, and synthetic DNA; and can be double-stranded or single-stranded,and if single stranded can be the coding strand or non-coding(anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:4, 10, 11, 41, 42, and 88-103. Also provided isa polynucleotide encoding a polypeptide having at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NOs: 4, 10, 11, 41, 42, and88-103.

The polynucleotides SEQ ID NOs: 5, 14, and 15 comprise the codingsequence for huMov19 variable domain heavy chain, variable domain lightchain version 1.00, and variable domain light chain version 1.60,respectively.

The invention further provides a polynucleotide comprising a sequenceselected from the group consisting of SEQ ID NOs:5, 14, 15, 37, 38, 43,44, 47, 48, and 120-127. Also provided is a polynucleotide having atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to SEQ ID NOs: 5, 14, 15,37, 38, 43, 44, 47, 48, and 120-127. Thus, in certain embodiments, thepolynucleotide comprises (a) a polynucleotide having at least about 95%sequence identity to SEQ ID NO:5, and/or (b) a polynucleotide having atleast about 95% sequence identity to SEQ ID NO:14 or 15. In certainembodiments, the polynucleotide comprises (a) a polynucleotide havingthe amino acid sequence of SEQ ID NO: 5; and/or (b) a polynucleotidehaving the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:15.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

V. METHODS OF USE AND PHARMACEUTICAL COMPOSITIONS

The FOLR1-binding agents (including antibodies, immunoconjugates, andpolypeptides) of the invention are useful in a variety of applicationsincluding, but not limited to, therapeutic treatment methods, such asthe treatment of cancer. In certain embodiments, the agents are usefulfor inhibiting tumor growth, inducing differentiation, reducing tumorvolume, and/or reducing the tumorigenicity of a tumor. The methods ofuse may be in vitro, ex vivo, or in vivo methods. In certainembodiments, the FOLR1-binding agent or antibody or immunoconjugate, orpolypeptide is an antagonist of the human FOLR1 to which it binds.

In one aspect, anti-FOLR1 antibodies and immunoconjugates of theinvention are useful for detecting the presence of FOLR1 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, such tissues includenormal and/or cancerous tissues that express FOLR1 at higher levelsrelative to other tissues. In certain embodiments, FOLR1 overexpressiondetects the presence of ovarian cancer, lung cancer, brain cancer,breast cancer, uterine cancer, renal cancer or pancreatic cancer.

In one aspect, the invention provides a method of detecting the presenceof FOLR1 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-FOLR1 antibodyunder conditions permissive for binding of the anti-FOLR1 antibody toFOLR1, and detecting whether a complex is formed between the anti-FOLR1antibody and FOLR1.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of FOLR1. In certain embodiments,the method comprises contacting a test cell with an anti-FOLR1 antibody;determining the level of expression (either quantitatively orqualitatively) of FOLR1 by the test cell by detecting binding of theanti-FOLR1 antibody to FOLR1; and comparing the level of expression ofFOLR1 by the test cell with the level of expression of FOLR1 by acontrol cell (e.g., a normal cell of the same tissue origin as the testcell or a cell that expresses FOLR1 at levels comparable to such anormal cell), wherein a higher level of expression of FOLR1 by the testcell as compared to the control cell indicates the presence of adisorder associated with increased expression of FOLR1. In certainembodiments, the test cell is obtained from an individual suspected ofhaving a disorder associated with increased expression of FOLR1. Incertain embodiments, the disorder is a cell proliferative disorder, suchas a cancer or a tumor.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-FOLR1antibody to FOLR1 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing FOLR1 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-FOLR1 antibody under conditions permissive for binding of theanti-FOLR1 antibody to FOLR1, and detecting whether a complex is formedbetween the anti-FOLR1 antibody and FOLR1 on the cell surface. Anexemplary assay for detecting binding of an anti-FOLR1 antibody to FOLR1expressed on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-FOLR1antibodies to FOLR1. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-FOLR1 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction.

In certain embodiments, anti-FOLR1 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-FOLR1antibody from any FOLR1 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-FOLR1antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-FOLR1 antibody afterformation of a complex between the anti-FOLR1 antibody and FOLR1, e.g.,by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-FOLR1 antibody.

In certain embodiments, the disease treated with the FOLR1-binding agentor antagonist (e.g., a huMov19 antibody or immunoconjugate) is a cancer.In certain embodiments, the cancer is characterized by tumors expressingfolate receptor 1 to which the FOLR1-binding agent (e.g., antibody)binds.

The present invention provides for methods of treating cancer comprisingadministering a therapeutically effective amount of a FOLR1-bindingagent to a subject (e.g., a subject in need of treatment). In certainembodiments, the cancer is a cancer selected from the group consistingof colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer,liver cancer, breast cancer, brain cancer, kidney cancer, prostatecancer, gastrointestinal cancer, melanoma, cervical cancer, bladdercancer, glioblastoma, and head and neck cancer. In certain embodiments,the cancer is ovarian cancer. In certain embodiments, the cancer is lungcancer. In certain embodiments, the subject is a human.

The present invention further provides methods for inhibiting tumorgrowth using the antibodies or other agents described herein. In certainembodiments, the method of inhibiting the tumor growth comprisescontacting the cell with a FOLR1-binding agent (e.g., antibody) invitro. For example, an immortalized cell line or a cancer cell line thatexpresses FOLR1 is cultured in medium to which is added the antibody orother agent to inhibit tumor growth. In some embodiments, tumor cellsare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and cultured in medium towhich is added an FOLR1-binding agent to inhibit tumor growth.

In some embodiments, the method of inhibiting tumor growth comprisescontacting the tumor or tumor cells with the FOLR1-binding agent (e.g.,antibody) in vivo. In certain embodiments, contacting a tumor or tumorcell with a FOLR1-binding agent is undertaken in an animal model. Forexample, FOLR1-binding agents can be administered to xenograftsexpressing one or more FOLR1s that have been grown in immunocompromisedmice (e.g. NOD/SCID mice) to inhibit tumor growth. In some embodiments,cancer stem cells are isolated from a patient sample such as, forexample, a tissue biopsy, pleural effusion, or blood sample and injectedinto immunocompromised mice that are then administered a FOLR1-bindingagent to inhibit tumor cell growth. In some embodiments, theFOLR1-binding agent is administered at the same time or shortly afterintroduction of tumorigenic cells into the animal to prevent tumorgrowth. In some embodiments, the FOLR1-binding agent is administered asa therapeutic after the tumorigenic cells have grown to a specifiedsize.

In certain embodiments, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of aFOLR1-binding agent. In certain embodiments, the subject is a human. Incertain embodiments, the subject has a tumor or has had a tumor removed.

In certain embodiments, the tumor expresses the folate receptor to whichthe FOLR1-binding agent or antibody binds. In certain embodiments, thetumor overexpresses the human FOLR1.

In certain embodiments, the tumor is a tumor selected from the groupconsisting of brain tumor, colorectal tumor, pancreatic tumor, lungtumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostatetumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris an ovarian tumor.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering atherapeutically effective amount of a FOLR1-binding agent to thesubject. In certain embodiments, the tumor comprises cancer stem cells.In certain embodiments, the frequency of cancer stem cells in the tumoris reduced by administration of the agent.

Thus, in certain embodiments the inventions provides methods of treatingcancer using huMov19 antibody and immunoconjugates. In certainembodiments, the huMov19 immunoconjugate is huMov19-SPDB-DM4;huMov119-sulfo-SPP-DM1; huMov19-SPP-DM1: or huMov19-PEG4-Mal-DM4.

The invention further provides methods of differentiating tumorigeniccells into non-tumorigenic cells comprising contacting the tumorigeniccells with a FOLR1-binding agent (for example, by administering theFOLR1-binding agent to a subject that has a tumor comprising thetumorigenic cells or that has had such a tumor removed. In certainembodiments, the tumorigenic cells are ovarian tumor cells.

The present invention further provides methods of reducingmyofibrolblast activation in the stroma of a solid tumor, comprisingcontacting the stroma with an effective amount of the FOLR1-bindingagent, polypeptide or antibody.

The present invention further provides pharmaceutical compositionscomprising one or more of the FOLR1-binding agents described herein. Incertain embodiments, the pharmaceutical compositions further comprise apharmaceutically acceptable vehicle. These pharmaceutical compositionsfind use in inhibiting tumor growth and treating cancer in humanpatients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g. carrier, excipient)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight polypeptides (e.g. less than about 10 amino acid residues);proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; carbohydratessuch as monosaccharides, disaccharides, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); and non-ionic surfactants such asTWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

An antibody or immunoconjugate of the invention can be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen preferably has complementary activities to the ADC of thecombination such that they do not adversely affect each other.Pharmaceutical compositions comprising the FOLR1-binding agent and thesecond anti-cancer agent are also provided.

For the treatment of the disease, the appropriate dosage of an antibodyor agent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antibody or agent is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g. reduction in tumor size). Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantibody or agent. The administering physician can easily determineoptimum dosages, dosing methodologies and repetition rates. In certainembodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, andcan be given once or more daily, weekly, monthly or yearly. In certainembodiments, the antibody or other FOLR1-binding agent is given onceevery two weeks or once every three weeks. In certain embodiments, thedosage of the antibody or other FOLR1-binding agent is from about 0.1 mgto about 20 mg per kg of body weight. The treating physician canestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues.

The combination therapy can provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

VI. KITS COMPRISING FOLR1-BINDING AGENTS

The present invention provides kits that comprise the antibodies,immunoconjugates or other agents described herein and that can be usedto perform the methods described herein. In certain embodiments, a kitcomprises at least one purified antibody against human folate receptor 1in one or more containers. In some embodiments, the kits contain all ofthe components necessary and/or sufficient to perform a detection assay,including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results. One skilledin the art will readily recognize that the disclosed antibodies,immunoconjugates or other agents of the present invention can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits comprising a FOLR1-binding agent (e.g., aFOLR1-binding antibody), as well as a second anti-cancer agent. Incertain embodiments, the second anti-cancer agent is a chemotherapeuticagent (e.g., gemcitabine or irinotecan).

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, can be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1 Chimerization of Murine Monoclonal Antibody Mov19

The variable region amino acid sequences for Mov19 were obtained fromthe NCBI database (accessions CAA68253 for the light chain (SEQ IDNO:24) and CAA68252 for the heavy chain (SEQ ID NO:23)) and thencodon-optimized and synthesized by Blue Heron Biotechnology. The lightchain variable region was cloned into the EcoRI and BsiWI sites of thepAbKZeo plasmid and the heavy chain variable region was cloned into theHindIII and Apa1 sites of the pAbG1Neo plasmid.

Example 2 Humanization of Murine Monoclonal Antibodies Mov19 and FR1-21

The Mov19 antibody was humanized following framework resurfacing methodspreviously described (Roguska M. et. al, Proc. Natl. Acad Sci. USA 1994February; 91:969-973) and (Roguska et al., Protein Eng. 9(10):895-904(1996)). Briefly, the average solvent accessibility for each variableregion framework residue was calculated using closely related solvedantibody structures from the PDB database, and positions with greaterthan a 30% average accessibility were marked as surface residues(Pedersen J. T. et. Al, J. Mol. Biol. 1994; 235: 959-973). The humansurface replacement sequence was selected by aligning the surfacepositions of murine antibody sequences with the corresponding positionsof the human antibody germline sequences in the Kabat database (Johnson,G. and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206). The mosthomologous human light chain variable region surface (clone DPK19, IMGTlocus IGKV2D-30*01 for Mov19 and IMGT locus IGKV1/OR2-0*01 for FR1-21)and the most homologous human heavy chain variable region surface (clone8M27, IMGT locus IGHV1-69*08 for Mov19 and IMGT locus IGHV5-51*02 forFR1-21) was selected to replace the murine Mov19 framework surfacepositions, leaving the 6 CDRs (Table 1) unaltered. The murine and humanMov19 and FR1-21 surface positions and residues are given in FIGS. 1A-D.

TABLE 1A The Mov19 and FR1-21 light and heavy chainCDRs as defined for resurfacing are provided.The Kabat definition for heavy chain CDR2is also given for both the murine and human antibodies. Mov19 CDRsFR1-21 CDRs Light Chain CDR1: KASQSVSFAGTSLMH CDR1: KASDHINNWLA(SEQ ID NO: 7) (SEQ ID NO: 27) CDR2: RASNLEA CDR2: GATSLET(SEQ ID NO: 8) (SEQ ID NO: 28) CDR3: QQSREYPYT CDR3: QQYWSTPFT(SEQ ID NO: 9) (SEQ ID NO: 29) Heavy Chain CDR1: GYFMN CDR1: SSYGMS(SEQ ID NO: 1) (SEQ ID NO: 30) CDR2 (AbM): RIHPYDGDTFCDR2 (AbM): TISSGGSYTY (SEQ ID NO: 131) (SEQ ID NO: 31) CDR3: YDGSRAMDYCDR3: DGEGGLYAMDY (SEQ ID NO: 3) (SEQ ID NO: 32) Kabat DefinedKabat Defined Mov19 HC CDR2 FR1-21 HC CDR2 Murine HC CDR2: RIHPYDGDTFYNQHC CDR2: TISSGGSYTYYP NFKD (SEQ ID NO: 128) DGVKG (SEQ ID NO: 33) HumanHC CDR2: RIHPYDGDTFYNQ HC CDR2: TISSGGSYTYYS KFQG(SEQ ID NO: 129)PGFQG (SEQ ID NO: 34)

None of the residue changes raised concerns for impacting theinteractions of either the Mov19 or FR1-21 CDRs with their targetepitopes on folate receptor 1, so no surface back mutations wereconsidered for the humanized sequences of either antibody. Theresurfaced Mov19 sequence did however introduce a consensus N-linkedglycosylation site at the light chain N74 (light chain version 1.00), soa second humanized light chain version was made to remove this site. Areview of the Kabat human light chain sequence database revealed thatthreonine is the most common residue found at light chain position 74 sothe humanized Mov19 light chain version 1.60 was built with a threonineat position 74. Position 74 is not a surface residue so this residuesubstitution has no impact on the humanization by resurfacing.Alignments of the variable region sequences of murine and humanizedMov19, and FR1-21 are given in FIG. 2.

The variable region sequences for humanized Mov19 and FR1-21 werecodon-optimized and synthesized by Blue Heron Biotechnology. Thesequences are flanked by restriction enzyme sites to facilitate cloningin-frame with the respective constant sequences in single chainmammalian expression plasmids. The light chain variable region wascloned into the EcoRI and BsiWI sites of the pAbKZeo plasmid. Theresulting plasmid DNAs encoding huMov19 light chain were deposited withthe ATCC as ATCC Deposit Nos. PTA-10773 and PTA-10774 and the resultingplasmid DNA encoding huFR1-21 light chain was deposited as ATCC DepositNo. PTA-10776. The heavy chain variable region was cloned into theHindIII and Apa1 sites of the pAbG1Neo plasmid. The resulting plasmidDNA encoding huMov19 heavy chain was deposited with the ATCC as ATCCDeposit No. PTA-10772 and the resulting plasmid DNA encoding huFR1-21heavy chain was deposited as ATCC Deposit No. PTA-10775. These plasmids,were then transfected as described in example 3 to produce huMov19. Theplasmid encoding either huMov19 light chain (i.e., that deposited asATCC Deposit No. PTA-10773 or PTA-10774) can be paired with the plasmidencoding huMov19 heavy chain to create a huMov19 antibody according tothe methods provided herein and as are well-known by one of ordinaryskill in the art.

Example 3 Recombinant Antibody Expression

The chimeric and humanized antibody constructs were transiently producedin either adherent HEK-293T cells using a standard calcium phosphateprocedure (BD Biosciences, CalPhos Mammalian Transfection Kit, Cat#631312) or in suspension adapted HEK-293T cells using a modified PEIprocedure [Durocher Y, Perret S, Kamen A High-level and high-throughputrecombinant protein production by transient transfection ofsuspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002 Jan.15; 30(2):E9] in spinner flasks. The PEI transient transfections wereperformed as previously described (Durocher, Y. et al., Nucleic AcidsRes. 30(2):E9 (2002)), except the HEK-293T cells were grown in Freestyle293 (Invitrogen) and the culture volume was left undiluted after theaddition of the PEI-DNA complexes. Both the adherent and suspensiontransient transfections were incubated for a week and then the clearedsupernatant was purified by a Protein A column followed by a CM columnion exchange chromatography as described below. As shown in FIG. 3,expression of huMov19 was at least 10-fold higher than expression ofchimeric Mov19 in transfected cells.

Example 4 Antibody Purification

Antibodies were purified from cleared cell culture supernatants usingstandard methods, such as, for example Protein A or G chromatography(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,supernatant was prepared for chromatography by the addition of 1/10volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant wasfiltered through a 0.22 μm filter membrane and loaded onto columnequilibrated with binding buffer (PBS, pH 7.3). The column was washedwith binding buffer until a stable baseline was obtained with noabsorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffercontaining 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.Fractions of approximately 0.25 mL were collected and neutralized by theaddition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) wasdialyzed overnight twice against 1×PBS and sterilized by filteringthrough a 0.2 μm filter membrane. Purified antibody was quantified byabsorbance at A₂₈₀.

Protein A purified fractions were further purified using ion exchangechromatography (IEX) with carboxymethyl (CM) chromatography. Briefly,samples from protein A purification were buffer exchanged into the startbuffer (10 mM potassium phosphate, 10 mM sodium chloride, pH 7.5) andfiltered through 0.22 μm filer. The prepared sample was then loaded ontoa CM fast flow resin (GE lifesciences) that was equilibrated with thestart buffer at a flow rate of 120 cm/hr. Column size was chosen to havesufficient capacity to bind all the antibody in the sample. The columnwas then washed with binding buffer until a stable baseline was obtainedwith no absorbance at 280 nm. Antibody was eluted by initiating agradient from 10 mM to 500 mM sodium chloride in 20 column volume (CV).Fractions with the UV reading above 50 mAu of the major peak werecollected. The purity (the percentage of monomer and soluble highmolecular weight aggregates) was assessed with size exclusionchromatography (SEC) on a TSK gel G3000SWXL, 7.8×300 mm with a SWXLguard column, 6.0×40 mm (Tosoh Bioscience, Montgomeryville, Pa.) usingan Agilent HPLC 1100 system (Agilent, Santa Clara, Calif.). Fractionswith desired purity (>95%) were pooled, buffer exchanged to PBS (pH 7.4)using TFF system, and sterilized by filtering through a 0.2 μm filtermembrane. Purified antibody was further tested for its purity by SEC andthe IgG concentration was determined by absorbance measurement at 280 nmusing an extinction coefficient of 1.47. Dilution was made if necessary.Alternatively, ceramic hydroxyapatite (CHT) can be used to polish bothmurine and humanized antibodies with improved selectivity. Type II CHTresin with 40 μm particle size (Bio-Rad Laboratories) was applied to thepolishing of antibodies with similar protocol as IEX chromatography. Thestart buffer for CHT was 20 mM sodium phosphate, pH 7.0 and antibody waseluted with a gradient of 20-160 mM sodium phosphate over 20 CV.

Example 5 Development of Murine Anti-FOLR1 Antibodies

There were two different immunization/screening series. First series hasled to generation of FR1-21 clone, second series has resulted ingeneration of FR1-48, FR1-49, FR1-57 and FR1-65 clones. In the firstseries mice were subcutaneously immunized with approximately 5×10⁶FOLR1-expressing KB cells (American Tissue Culture Collection, ATCCCCL-17). In the second series 300-19 cells expressing human FOLR1 ontheir surface were used to immunize mice. To make these cells, the humanFOLR1 amino acid sequence was obtained from the NCBI website (accessionNP_(—)057937), then it was codon optimized and synthesized by Blue Heronbiotechnologies, flanked by EcoRI and Xba1 restriction sites tofacilitate cloning into the pSRa mammalian expression vector. 300-19cells, a pre-B cell line derived from a Balb/c mouse (Reth et al.,Nature, 317:353-355 (1985)), were transfected with the pSRa-FolR1expression plasmid to stably express high levels of human FOLR1 on thecell surface. Standard immunization protocols known to those of skill,for example, such as those used at ImmunoGen, Inc were applied for bothseries. Immunized mice were boosted with antigen three days before beingsacrificed for hybridoma generation. Spleens from mice was collectedaccording to standard animal protocols, such as, for example grindingtissue between two sterile, frosted microscopic slides to obtain asingle cell suspension in RPMI-1640 medium. The spleen cells werecentrifuged, pelleted, washed, and fused with a murine myeloma, such as,for example P3X63Ag8.653 cells (Kearney et al., J. Immunol.,123:1548-1550 (1979)) using polyethylene glycol-1500 (Roche 783 641).The fused cells were resuspended in RPMI-1640 selection mediumcontaining hypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262) andselected for growth in 96-well flat-bottomed culture plates(Corning-Costar 3596, 0.2 ml of cell suspension per well) at 37° C. with5% CO₂. After 5 days of incubation, 0.1 ml of culture supernatant wereremoved from each well and replaced with 0.1 ml of RPMI-1640 mediumcontaining hypoxanthine-thymidine (HT) supplement (Sigma H-0137).Incubation at 37° C. with 5% CO₂ was continued until hydridoma cloneswere ready for antibody screening. Other techniques of immunization andhybridoma production can also be used, including those described inLangone et al. (Eds., “Immunochemical Techniques, Part I”, Methods inEnzymology, Academic Press, volume 121, Florida) and Harlow et al.(“Antibodies: A Laboratory Manual”; Cold Spring Harbor Laboratory Press,New York (1988)).

TABLE 1B The FR1-48, 49, 57, and 65 light andheavy chain CDRs are provided. The Kabatdefinition for heavy chain CDR2 is also given for both the murine and human antibodies. FR1-48 CDRs FR1-49 CDRsFR1-57 CDRs FR1-65 CDRs Light Chain CDR1- CDR1- CDR1- CDR1- RASENIYSNLARASENIYTNLA RASQNINNNLH KASQNVGPNVA (SEQ ID NO: 57) (SEQ ID NO: 63)(SEQ ID NO: 69) (SEQ ID NO: 75) CDR2- CDR2- CDR2- CDR2- AATNLAD TASNLADYVSQSVS SASYRYS (SEQ ID NO: 58) (SEQ ID NO: 64) (SEQ ID NO: 70)(SEQ ID NO: 76) CDR3- CDR3- CDR3- CDR3- QHFWASPYT QHFWVSPYT QQSNSWPHYTQQYNSYPYT (SEQ ID NO: 59) (SEQ ID NO: 65) (SEQ ID NO: 71)(SEQ ID NO: 77) Heavy Chain CDR1- CDR1- CDR1- CDR1- TNYWMQ TNYWMY SSFGMHTSYTMH (SEQ ID NO: 60) (SEQ ID NO: 66) (SEQ ID NO: 72) (SEQ ID NO: 78)CDR2- CDR2- CDR2- CDR2- AIYPGNGDSR AIYPGNSDTT YISSGSSTIS YINPISGYTN(SEQ ID NO: 61) (SEQ ID NO: 67) (SEQ ID NO: 73) (SEQ ID NO: 79) CDR3-CDR3- CDR3- CDR3- RDGNYAAY RHDYGAMDY EAYGSSMEY GGAYGRKPMDY(SEQ ID NO: 62) (SEQ ID NO: 68) (SEQ ID NO: 74) (SEQ ID NO: 80)Kabat HC CDR2 Murine Murine Murine Murine AIYPGNGDSRYTQKFAIYPGNSDTTYNLKFK YISSGSSTISYADIV YINPISGYTNYNQKF KG G KG KD(SEQ ID NO: 81) (SEQ ID NO: 130) (SEQ ID NO: 84) (SEQ ID NO: 86) HumanHuman Human Human AIYPGNGDSRYTQKF AIYPGNSDTTYNQKFQ YISSGSSTISYADSVYINPISGYINYNQKF QG G KG QG (SEQ ID NO: 82) (SEQ ID NO: 83)(SEQ ID NO: 85) (SEQ ID NO: 87)

Example 6 Hybridoma Screening and Selection

FOLR1-300-19 cells transfected with human FOLR1 and KB cells were usedin the first and second series of screenings correspondently. Culturesupernatants from the hybridoma were screened by flow cytometry forsecretion of mouse monoclonal antibodies that bind to FOLR1 positivecells, such as FOLR1-expressing 300-19 cells or KB cells, but not to theFOLR1 negative cells, such as non-transfected 300-19 cells. 0.1 ml ofhybridoma supernatants was incubated for 3 h with either FOLR1-positivecells or the non-transfected 300-19 cells (1×10⁵ cells per sample) in0.1 ml FACS buffer (RPMI-1640 medium supplemented with 2% normal goatserum). Then, the cells were centrifuged, pelleted, washed, andincubated for 1 hour with 0.1 ml of PE-conjugated goat anti mouseIgG-antibody (such as obtainable from, for example Jackson Laboratory, 6μg/mL in FACS buffer). The cells were centrifuged, pelleted again,washed with FACS buffer and resuspended in 0.2 ml of PBS containing 1%formaldehyde. Cell-associated fluorescence was measured using aFACSCalibur flow cytometer with the HTS multiwell sampler or a FACSarray flow cytometer and analyzed using CellQuest Pro (all from BDBiosciences, San Diego, US). Positive hybridoma clones were subcloned bylimiting dilution. One subclone from each hybridoma, which showed thesame reactivity against FOLR1 as the parental cells by flow cytometry,was chosen for subsequent analysis. Stable subclones were cultured andthe isotype of each secreted anti-FOLR1 antibody was identified usingcommercial isotyping reagents (Roche 1493027). Murine antibodies wereprotein A purified from cleared hybridoma media as described above.These antibodies were designated FR-1 antibodies.

Example 7 Murine Monoclonal Antibody Purification

Antibodies were purified from hybridoma subclone supernatants usingstandard methods, such as, for example Protein A or G chromatography(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,supernatant was prepared for chromatography by the addition of 1/10volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant wasfiltered through a 0.22 μm filter membrane and loaded onto columnequilibrated with binding buffer (PBS, pH 7.3). The column was washedwith binding buffer until a stable baseline was obtained with noabsorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffercontaining 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.Fractions of approximately 0.25 mL were collected and neutralized by theaddition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) wasdialyzed overnight twice against 1×PBS and sterilized by filteringthrough a 0.2 μm filter membrane. Purified antibody was quantified byabsorbance at A₂₈₀.

Example 8 Binding Characterization by Flow Cytometry

Binding specificity was tested by flow cytometry using purifiedantibodies. FACS histograms demonstrating the binding of anti-FOLR1 toFOLR1-expressing 300-19 cells and the absence of binding to the parental300-19 cells are shown in FIG. 4. Each antibody was incubated for 3hours with either FOLR1-expressing 300-19 cells or the non-transfected300-19 cells (1×10⁵ cells per sample) in 0.1 ml FACS buffer (RPMI-1640medium supplemented with 2% normal goat serum). Then, the cells werepelleted, washed, and incubated for 1 hour with 0.1 ml ofFITC-conjugated goat anti-mouse IgG-antibody (such as is obtainablefrom, for example Jackson Laboratory, 6 μg/mL in FACS buffer). The cellswere pelleted again, washed with FACS buffer and resuspended in 200 μLof PBS containing 1% formaldehyde. Samples were acquired using aFACSCalibur flow cytometer with the HTS multiwell sampler or a FACSarray flow cytometer and analyzed using CellQuest Pro (all from BDBiosciences, San Diego, US). The FACS histograms of anti-FOLR1antibodies showed a fluorescence shift, while parental 300-19 cells didnot. Also, no significant fluorescence shift was detected when either ofthe cell lines was incubated only with FITC conjugated goat anti-humanIgG-antibody alone.

Example 9 Cloning and Sequencing of the VL and VH Regions of muFR1-21

Total cellular RNA was prepared from 5×10⁶ hybridoma cells using anRNeasy kit (QIAgen) according to the manufacturer's protocol. cDNA wassubsequently synthesized from total RNA using the SuperScript II cDNAsynthesis kit (Invitrogen). The procedure for the first round degeneratePCR reaction on the cDNA derived from hybridoma cells was based onmethods described in Wang et al. ((2000) J Immunol Methods. January 13;233(1-2):167-77) and Co et al. ((1992) J Immunol. February 15;148(4):1149-54). VH sequences were amplified by PCR using the followingdegenerate primers: EcoMH1 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ IDNO:50) EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO:51) andBamIgG1 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO:52). VLsequences were amplified by PCR using the following degenerate primers:SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO:53) and HindKLTATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQ ID NO:54). (Mixedbases are defined as follows: N=G+A+T+C, S=G+C, Y=C+T, M=A+C, R=A+G,W=A+T).

The PCR reaction mixtures were then run on a 1% low melt agarose gel,the 300 to 400 bp bands were excised, purified using Zymo DNA minicolumns, and sent to Agencourt Biosciences for sequencing. Therespective 5′ and 3′ PCR primers were used as sequencing primers togenerate the variable region cDNAs from both directions. The amino acidsequences of VH and VL regions were obtained by translating the DNAsequencing results with VectorNTI software.

To identify 5′end primer sequencing artifacts in the preliminaryvariable region cDNA sequences, the NCBI IgBlast site was utilized tosearch for the murine germline sequences from which the antibodysequences were derived. The cleaned up variable region sequences werethen combined with the NCBI reference sequences for the specificantibody constant regions to assemble expected full length murineantibody sequences. The molecular weight of the expected murine Fr1-21light and heavy chains were then calculated and compared with the massmeasured by liquid chromatography/mass spectrophotometric analysis(LC/MS). The murine FR1-21 heavy chain matched the measured mass, butthe light chain required a follow up sequencing effort to determine the5′ end sequence. The CD37-1LClead1 PCR primerttttgaattcgccaccatgaagtttccttctcaacttct (SEQ ID NO:55) was designed toanneal to the germline linked leader sequence of the murine antibody sothat this new PCR reaction would yield a complete variable region cDNAsequence, unaltered by the primers. The PCR reactions, bandpurifications, and sequencing were performed as described above and thenew complete sequence encoded a light chain that matched the Fr1-21light chain mass measured by LC/MS.

Example 10 Expression of Reference Antibodies

The Morphotech anti-FOLR1 antibody, MorAb-003 (Farletuzumab), amino acidsequence was obtained from the World Health Organization (WHO)International Nonproprietary Names for Pharmaceutical Substances (INN)list and was codon-optimized and synthesized by Blue HeronBiotechnology. The light chain variable region sequence is flanked byEcoRI and BsiWI restriction enzyme sites and the heavy chain variableregion sequence flanked by HindIII and Apa1 restriction enzyme sites forcloning in-frame with the respective constant sequences in single chainmammalian expression plasmids. Cloning, expression and purification wascarried out as described for humanized Mov19 and Fr1-21 above.

Example 11 ADCC Activity of huMov19

A lactate dehydrogenase (LDH) release assay was used to measureantibody-dependent cell mediated cytotoxicity (ADCC) of tumor cellslines using freshly isolated human natural killer (NK) cells as effectorcells (e.g., Shields, J. Biol. Chem., 276(9):6591-6604 (2001)). NK cellswere first isolated from human blood from a normal donor (Research BloodComponents, Inc., Brighton, Mass.) using a modified protocol for the NKIsolation Kit II (Miltenyi Biotech, 130-091-152). Blood was diluted2-fold with 1×PBS. 25 mL of diluted blood was carefully layered over 25mL of Ficoll Paque in a 50 mL conical tube and centrifuged at 400 g for45 min at RT. The peripheral blood mononuclear cells (PBMC) werecollected from the interface, transferred into a new conical 50 mL tube,and washed once with 1×PBS. The PBMC were resuspended in 2 mL ofNK-isolation buffer (1×PBS, 0.5% BSA, 2 mM EDTA), and then 500 CpL ofBiotin-Antibody Cocktail were added to the cell suspension. TheBiotin-Antibody Cocktail contains biotinylated antibodies that bind tothe lymphocytes, except for NK cells, resulting in a negative selectionof NK cells. The mixture was incubated at 4° C. for 10 minutes, and then1.5 mL of NK-isolation buffer and 1 mL of Anti-Biotin Micro Beads wereadded. The cell antibody mixture was incubated for another 15 minutes at4° C. Next, cells were washed once with 50 mL of NK-isolation buffer andresuspended in 3 mL of NK-isolation buffer. Then, a MACS LS column wasmounted on the autoMACS separator (Miltenyi Biotech) and pre-washed with3 mL of NK-isolation Buffer. The cell suspension was automaticallyapplied onto the column, washed and the effluent fraction with unlabeledNK cells was collected into a new 50 mL conical tube. The resulting NKcells were plated into 30 mL of complete RPMI media (RPMI-1640supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, 1mM HEPES, 1 mM Sodium Pyruvate, 1% 100×MEM non-essential Amino AcidSolution) overnight. The subsequent assay and all dilutions were carriedout in RHBP medium (RPMI 1640 medium supplemented with 20 mM HEPES, pH7.4, 0.1% BSA and 1% penicillin streptomycin). Various concentrations ofantibodies in RHBP medium were aliquoted in duplicate at 50 μL/well intoa round bottom 96-well plate. The target cells were resuspended at 10⁶cells/mL in RHBP medium and added at 100 μL/well to each well containingantibody dilutions. The plate containing target cells and antibodydilutions was incubated for 30 minutes at 37° C. NK cells were thenadded to the wells containing the target cells at 50 μL/well. Thetypical ratio was about 1 target cell to 3-4 NK cells. At least thefollowing controls were set up for each experiment: NK cells alone,target cells alone (spontaneous LDH release), target cells with NK cells(antibody independent LDH release), target cells with 10% TritonX-100(maximum LDH release). The mixtures were incubated at 37° C. for 4 hoursto allow for cell lysis. Plates were centrifuged for 10 minutes at 1200rpm, and 100 μL of the supernatant was carefully transferred to a newflat bottom 96-well plate. LDH reaction mixture (100 μL/well) from theCytotoxicity Detection Kit (Roche 1 644 793) was added to each well andincubated at room temperature for 5 to 30 min. The optical density ofsamples was measured at 490 nm (OD₄₉₀). The percent specific lysis ofeach sample was determined using the following formula: percent specificlysis=(sample value−spontaneous release)/(maximum release−spontaneousrelease)*100.

Incubation with huMov19 lead to good ADCC activity against IGROV-1 cellsin the presence of human NK effector cells. ADCC activity on IGROV-1cells was compared for huMov19, huFR-1-21, Mor003, and chTK1 (isotypecontrol) (FIG. 6). Treatment with 0.9 ng/ml huMov19 resulted inapproximately 30% IGROV-1 cell lysis, similar to activity that wasobserved with the other anti-FOLR1 antibodies. ADCC activity by huMov19had an EC₅₀ of 0.20 ng/mL, huFr-1-21 had an EC₅₀ of 0.11 ng/mL, Mor003of 0.16 ng/mL and chTK1 did not show any activity against IGROV-1 cells.

Example 12 Preparation of Anti-FOLR1 Immunoconjugates

Preparation of huMOV19v1.6-sulfo-SPDB-DM4

The exemplary 2-sulfo-SPDB linker was dissolved in DMA. The huMOV19v1.6antibody was incubated at 8 mg/mL with a 12 fold molar excess of2-sulfo-SPDB linker for approximately 2 hours at 25° C. at pH 7.5. Thereaction mixture was purified using a SEPHADEX™ G25F column equilibratedwith 50 mM potassium phosphate buffer containing 50 mM NaCl, 2 mM EDTA,pH 6.5. The maytansinoid DM4 was dissolved in dimethylacetamide (DMA,final concentration is 5%) and a 1.7 fold molar excess compared to thelinker was added drop wise to the sulfo-SPDB modified antibody. Thereaction mixture was adjusted to pH 7.5 with 1 m HEPES buffer. Afterovernight incubation at room temperature, the conjugated antibody waspurified by chromatography on SEPHADEX™ G25F equilibrated with 10 mMhistidine, 250 mM glycine, 1% sucrose, pH 5.5 The number of DM4molecules linked per antibody molecule was determined using thepreviously reported extinction coefficients for antibody andmaytansinoid (Widdison, W C, et al. J Med Chem, 49:4392-4408 (2006)).The percentage of total free maytansinoid species were determined asdescribed above. Conjugates with 3.5-4 DM4 molecules per huMov19v1.6antibody were obtained with <1% present as unconjugated maytansinoid.

Preparation of huMOV19v1.6-SPP-DM1

The exemplary N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linkerwas dissolved in ethanol. The huMOV19v1.6 antibody was incubated at 8mg/mL with a 6.5 to 6-fold molar excess of SPP linker for approximately2 hours at room temperature in 50 mM potassium phosphate buffer (pH 6.5)containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The SPP modifiedantibody was diluted 2-fold in PBS, pH 6.5 and modified with a 1.5 foldmolar excess of the maytansinoid DM1 by the addition of a concentratedsolution (15-30 mM) of DM1 in dimethylacetamide (DMA). The concentrationof DMA was adjusted to 5% and after overnight incubation at roomtemperature, the conjugated antibody was purified by chromatography onSEPHADEX™ G25F equilibrated 10 mM, 250 mM glycine, 1% sucrose pH 5.5.The number of DM1 molecules linked per antibody molecule was determinedusing the previously reported extinction coefficients for antibody andDM1 (Liu et al., Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). Thepercentage of free maytansinoid present after the conjugation reactionwas determined by injecting 20-50 g conjugate onto a HiSep™ columnequilibrated in 25% acetonitrile in 100 mM ammonium acetate buffer, pH7.0, and eluting in acetonitrile. The peak area of total freemaytansinoid species (eluted in the gradient and identified bycomparison of elution time with known standards) was measured using anabsorbance detector set to a wavelength of 252 nm and compared with thepeak area related to bound maytansinoid (eluted in the conjugate peak inthe column flow-through fractions) to calculate the percentage of totalfree maytansinoid species. Conjugates with 3.5-4 DM1 molecules perhuMOV19v1.6 were obtained with <1% present as unconjugated maytansinoid.

Preparation of huMOV19v1.6 SPDB-DM4

The exemplary N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) linkerwas dissolved in ethanol. The huMOV19v1.6 antibody was incubated at 8mg/mL with a 5.5-5 fold molar excess of SPDB linker for approximately 2hours at room temperature in 50 mM potassium phosphate buffer (pH 6.5)containing 50 mM NaCl, 2 mM EDTA, and 3% ethanol. The SPDB modifiedantibody was diluted 2-fold in PBS, pH 6.5 and modified with a 1.5 foldmolar excess of the maytansinoid DM4 by the addition of a concentratedsolution (15-30 mM) of DM4 in dimethylacetamide (DMA). After overnightincubation at room temperature, the conjugated antibody was purified bychromatography on SEPHADEX™ G25F equilibrated with 10 mM histidine, 250mM glycine, 1% sucrose pH 5.5. The number of DM4 molecules linked perantibody molecule was determined using the previously reportedextinction coefficients for antibody and maytansinoid (Widdison, W C, etal. J Med Chem, 49:4392-4408 (2006)). The percentage of total freemaytansinoid species were determined as described above. Conjugates with3.5-4 DM4 molecules per huMOV19v1.6 antibody were obtained with <1%present as unconjugated maytansinoid.

Preparation of huMOV19v1.0-3-sulfo-mal-DM4

The NHS-3-sulfo-mal linker and DM4 were dissolved separately in DMA. Thelinker and DM4 thiol were mixed together in a solution of DMA containing40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a molar ratio ofDM4 to linker of 1.6:1 and a final concentration of DM4 equal to 10 mM.The mixture was reacted for 2 hours at 25 C. Without purification, thereaction mixture was added so that an equivalent of 9.6 molar excess oflinker to antibody was added to a solution of huMOV19v1.0 antibody inphosphate buffer (pH7.5) under final conjugation conditions of 4 mg/mLantibody, 90% phosphate buffer/10% DMA pH7.5 (v/v). After an overnightincubation at room temperature, the conjugation mixture was purified bychromatography on SEPHADEX G25 equilibrated in PBS pH7.5. ThehuMOV19v1.0-3-sulfo-mal-DM4 was then dialyzed into a buffer containing9.55 mM Phosphate, 139.6 mM NaCl, pH6.5. The number of DM4 moleculeslinked per antibody molecule was determined using the previouslyreported extinction coefficients for antibody and maytansinoid(Widdison, W C, et al. J Med Chem, 49:4392-4408 (2006)). The percentageof total free maytansinoid species was determined as described above.Conjugates with 3.5-4 DM4 molecules per huMOV19v1.0 antibody wereobtained with <1% present as unconjugated maytansinoid.

Preparation of huMOV19v1.0-SMCC-DM1

The NHS-sulfo-SMCC linker and DM1 were dissolved separately in DMA. Thelinker and DM1 thiol were mixed together in a solution of DMA containing40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a molar ratio ofDM1 to linker of 1.2:1 and a final concentration of DM1 equal to 3.75mM. The mixture was reacted for 75 minutes at 20° C. Withoutpurification, the reaction mixture was added so that an equivalent of6.4 molar excess of linker to antibody was added to a solution ofhuMOV19v1.0 antibody in phosphate buffer (pH7.5) under final conjugationconditions of 4 mg/mL antibody, 88% 50 mM Potassium Phosphate, 50 mMNaCl, 2 mM EDTA, pH 7.5/12% DMA pH7.5 (v/v). After 2 hour incubation at20° C., the conjugation mixture was purified by chromatography onSEPHADEX G25 equilibrated in PBS pH7.5. The huMOV19v1.0-SMCC-DM1 wasthen dialyzed into a buffer containing 250 mM Glycine, 10 mM HistidinepH5.5. The number of DM1 molecules linked per antibody molecule wasdetermined using the previously reported extinction coefficients forantibody and maytansinoid (Widdison, W C, et al. J Med Chem,49:4392-4408 (2006)). The percentage of total free maytansinoid specieswas determined as described above. Conjugates with 3.5-4 DM1 moleculesper huMOV19v1.0 antibody were obtained with <2.8% present asunconjugated maytansinoid.

Preparation of huMOV19v1.0-PEG4-mal-DM1

The NHS-PEG4-mal-DM1 1 step reagent was dissolved in DMA. ThehuMov19v1.0 antibody was incubated at 5 mg/mL with a 5.7 fold molarexcess of NHS-PEG4-mal-DM1 overnight at 25° C. in 50 mM KPi, 50 mM NaCl,2 mM EDTA, pH 7.5 and 10% DMA by volume. The reaction mixture waspurified by SEPHADEX G25 column equilibrated in PBS pH7.5. ThehuMOV19v1.0-PEG4-mal-DM1 was dialyzed into buffer containing 250 mMGlycine, 10 mM Histidine pH5.5. The number of DM1 molecules linked perantibody molecule was determined using the previously reportedextinction coefficients for antibody and maytansinoid (Widdison, W C, etal. J Med Chem, 49:4392-4408 (2006)). The percentage of total freemaytansinoid species was determined as described above. Conjugates with3.5-4 DM1 molecules per huMOV19v1.0 antibody were obtained with <1.1%present as unconjugated maytansinoid.

Example 13 Binding Affinity of Antibodies and Conjugates

Binding affinities of anti-FOLR1 antibodies and of their SPDB-DM4,PEG4Mal-DM4, SMCC-DM1, or anti-FOLR1-sulfo-SPDB-DM4 conjugates wereassayed by Flow Cytometry. FOLR1-expressing SKOV3 cells were incubatedwith varying concentrations of anti-FOLR1 antibodies or their conjugatesand processed as described above for flow cytometry analysis. Dataanalysis was performed using CellQuest Pro (BD Biosciences, San Diego,US) and for each sample the mean fluorescence intensity for FL1 (MFI)was exported and plotted against the antibody concentration in asemi-log plot. A dose-response curve was generated by non-linearregression and the value for the apparent equilibrium dissociationconstant (K_(d)) of the test-samples for the binding to SKOV3 cells wascalculated using GraphPad Prism v4 (GraphPad software, San Diego,Calif.) and presented in FIG. 5. The results demonstrate thatconjugation to either DM1 or DM4 through either of the linkers used, didnot notably alter the affinity of either of the antibodies (e.g.,huMov19).

Example 14 In Vitro Cytotoxicity Assays

The ability of exemplary muFR1-9, muFR-1-13, muFR-1-22, muFR1-23,huFR1-23, muFR1-21, and huFR1-21 conjugates to inhibit cell growth wasmeasured using in vitro cytotoxicity assays by the method described inKovtun Y V et al. (Cancer Res 66: 3214-3221 (2006)). A PEG4-mal-DM4conjugate in various concentrations was added to FOLR1-expressing KBcells in a 96 well plate at 1,000 cells per well in 100 μL in completeRPMI medium (RPMI-1640, 10% fetal bovine serum, 2 mM glutamine, 1%gentamycin, all reagents from Invitrogen). Antibodies and conjugateswere diluted into complete RPMI medium using 3-fold dilution series and100 μL were added per well. The final concentration typically rangedfrom 3×10⁻⁸ M to 4.6×10⁻¹² M. Control wells containing cells and themedium but lacking the conjugates, and wells containing medium only wereincluded in each assay plate. The plates were incubated from four to sixdays at 37° C. in a humidified atmosphere containing 5% CO₂. WST-8reagent, 10% v/v (Dojindo Molecular Technologies, Gaithersburg, Md., US)was then added to the wells and the plates were incubated at 37° C. for2-6 h. WST-8 is reduced by dehydrogenases in living cells to an orange(maximum formazan product that is soluble in tissue culture medium. Theamount of formazan produced is directly proportional to the number ofliving cells. Plates were analyzed by measuring the absorbance at 450 nm(A₄₅₀) and at and 650 nm (A₆₅₀) in a multiwell plate reader. First, thebackground of cells' opalescence (A₆₅₀) was subtracted from A₆₅₀. Theresulting A*₄₅₀ was then used to determine the surviving fraction ofcells. Background A*₄₅₀ absorbance was that of wells with medium andWST-8 only. The surviving fraction was calculated as follows: Percentviability=100×(A*₄₅₀ treated sample−A*₄₅₀ background)/(A*₄₅₀ untreatedsample−A*₄₅₀ background). The surviving fraction values were plottedagainst antibody or conjugate concentration in a semi-log plot for eachtreatment. From these data IC₅₀ values were then determined usingGraphPad Prism v4 (GraphPad software, San Diego, Calif.) and presentedin FIG. 5. The results shown in FIG. 5 demonstrate that all conjugatesare similarly active in their cytotoxic potency against FOLR1-expressingKB cells. To further verify the specificity of theanti-FOLR1-maytansinoid conjugates towards FOLR1, their activities wereevaluated in the presence of an excess of non-conjugated antibodiesagainst KB cells. Addition of an excess of competing non-conjugatedantibody to the conjugates suppressed their cytotoxicity, as seen inFIG. 7. These data indicate that the conjugates kill KB cells in anantigen-dependent manner. Additional data demonstrated thathuMov19-SPDB-DM4 induced cell cycle arrest in the G2/M phase in KB cellsin in vitro assays.

Example 15 In Vivo Efficacy of huMov19-PEG4Mal-DM4 and huMov19-SPDB-DM4Conjugates in Comparison with Similar Non-Targeting Conjugates in a KBXenograft Model

FOLR1-targeting cleavable conjugate huMov19-SPDB-DM4 in comparison withnon-targeting huC242-SPDB-DM4, and non-cleavable conjugatehuMov19-PEG4-Mal-DM4 in comparison with non-targeting huC242-PEG4Mal-DM4were tested using an established xenograft model of KB cells implantedsubcutaneous into SCID mice. Mice were randomized by body weight intotreatment groups and treated either singly (SPDB conjugates) on day 3post cell inoculation, or three times weekly on days 3, 10, and 17 postcell inoculation with 5 and 10 mg/kg of a conjugate, respectively. Themedian tumor volume of the different treatment groups is plotted in FIG.8. The treatments with either huMov19-SPDB-DM4, or huMov19-PEG4Mal-DM4resulted in a decrease in median tumor volume as compared to the PBScontrol, while the treatments with either of the respectivenon-targeting conjugate did not produce any significant effect.

Example 16 In Vivo Efficacy of Anti-FOLR1-PEG4Mal-DM4 Conjugates in a KBXenograft Model

PEG4Mal-DM4 conjugates of the exemplary anti-FOLR1 antibodies huMov19,muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 were testedusing an established xenograft model of KB cells implanted subcutaneousinto SCID mice. Mice were randomized by body weight into treatmentgroups and treated once on day 3 post cell inoculation with 10 mg/kg ofone of the conjugates listed above or with PBS only. HuMov19-PEG4Mal-DM4was shown above to be similar to PEG4Mal-DM4 conjugates of muFR-1-9,muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 in its cytotoxic potencyin vitro. HuMov19-PEG4Mal-DM4 and huFR-1-21-PEG4Mal-DM4 weresignificantly more potent in vivo than any of the other conjugates,resulting in a more pronounced decrease in median tumor volume (FIGS. 9and 10). The potency was also demonstrated to be dose-dependent (FIG.11) and choice of linker played a role as well (FIGS. 12 and 13).

Example 17 In Vivo Efficacy of Anti-FOLR1-Sulfo-SPDB-DM4 Conjugates in aXenograft Models

Anti-FOLR1 huMov19-sulfo-SPDB-DM4 conjugates were tested in threeovarian serous adenocarcinoma xenografts: OVCAR-3, IGROV-1, and OV-90.Each of these xenograft tumors showed FOLR1 expression levels comparableto patient tumors when measured using a calibrated immunohistochemical(IHC) staining method on formalin-fixed paraffin-embedded sections. Micebearing established subcutaneous xenograft tumors (approximately 100mm³) were treated with a single intravenous injection ofhuMov19-sulfo-SPDB-DM4 conjugate at 1.2, 2.5, and 5.0 mg/kg (based onantibody concentration; FIGS. 14-16 show the concentration of themaytansanoid conjugate in μg/kg). The conjugate was active in all threemodels evaluated. In OVCAR-3 xenografts, the minimally efficacious dose(MED) was 1.2 mg/kg (FIG. 14). The higher dose levels were highlyactive, resulting in complete regressions (CR) in 4/6 and 2/6 mice inthe 2.5 and 5.0 mg/kg treatment groups, respectively. Treatment with theconjugate resulted in strong anti-tumor activity in both IGROV-1 andOV-90 xenograft models, with a MED of 2.5 mg/kg, single injection (FIGS.15 and 16). These data demonstrate the strong anti-tumor activity ofhuMov19-sulfo-SPDB-DM4 conjugates against ovarian xenograft tumors withFOLR1 expression levels comparable to patient tumors.

Example 18 Effect of Linkers on Immunoconjugate Efficacy

The anti-FOLR1 antibody huMov19 was linked to DM1 or DM4 via thedisulfide-containing cleavable linkers SPP, SPDB, or sulfo-SPDB, or viathe non-cleavable linker SMCC. The in vitro cytotoxic activities ofthese conjugates on KB, IGROV-1 and JEG-3 cell lines was examined. FACSanalysis indicated that the KB (cervical) cells had >2,000,000 antibodybinding sites per cell. The IGROV-1 (ovarian) cells had 260,000 antibodybinding sites per cell, and the JEG-3 (choriocarcinoma) cells had 40,000antibody binding sites per cell. The results of the in vitrocytotoxicity are summarized in Table 2 below. The cleavable conjugatesdisplayed markedly greater in vitro activities compared with those ofthe SMCC-conjugate.

TABLE 2 Effect of immunoconjugate linkers on cytotoxicity in vitro.IC₅₀, nM (n = 3), Ab-based Cells SPP-DM1 SPDB-DM4 Sulfo-SPDM-DM4SMCC-DM1 KB 0.1 0.1 0.1 0.1 Igrov 1 0.1 0.1 0.3 1.0 Jeg3 0.2 0.2 3.0 20

The in vivo activities of the conjugates in FOLR-positive KB- andOVCAR-3-tumor models were also tested. The results shown in FIG. 17demonstrate that cleavable SPDB-DM4 and sulfo-SPDB-DM4 conjugates aremore patent than non-cleavable SMCC-DM1 conjugates in vivo. In addition,among the cleavable conjugates, the SPP-DM1 conjugate was less activethan either the SPDB-DM4 or sulfo-SPDB-DM4 conjugates in both xenograftmodels (FIG. 18). The two latter conjugates were similarly activeagainst KB tumors, whereas the sulfo-SPDB-DM4 conjugate was more activeagainst the OVCAR-3 model. The data obtained using the OVCAR-3 model issummarized in Table 3 below.

TABLE 3 Effect of immunoconjugate linkers on tumor size in OVCAR-3xenograft model. Tumor over Partial Complete Conjugate control (%)Response Response Response SPP-DM1 54 0/6 0/6 Inactive SPDB-DM4  9 6/61/6 Highly active Sulfo-DPDB-  0 6/6 4/6 Highly active DM4

These data demonstrate that immunoconjugates containing a cleavablelinker show increased efficacy both in vitro and in vivo, and anti-FOLR1immunoconjugates containing sulfo-SPDB are highly active in tumormodels.

Example 19 In Vitro and In Vivo Efficacy of huFR1 Antibody SMCC-DM1Conjugate

Anti-FOLR1 huFR1-48, huFR1-49, huFR1-57, and huFR1-65 were conjugatedwith SMCC linker and DM1 and the effects on KB cells, and in vivo usingthe above-described xenograft models were analyzed as described above.While each of the antibodies showed similar efficacy in the KB cellmodel, the huFR1-48, huFR1-49, huFR1-57, and huFR1-65 immunoconjugatesshowed variable, but significant, in vivo efficacy at a 200 μg/kg dosein a xenograft model system (Table 4 and FIG. 19).

TABLE 4 In vitro and in vivo efficacy of huFR1 antibody SMCC-DM1conjugate Apparent huAb-smcc-DM1 affinity activity on KB inhuAb-smcc-DM1 Clone # (nM) vitro (nM) activity in vivo huFR1-48 0.130.05 + huFR1-49 0.08 0.10 + huFR1-57 0.14 0.10 + huFR1-65 0.15 0.10 +huMov19 0.06 0.10 ++

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

SEQUENCES

huMov19 vHC CDR1 SEQ ID NO: 1 GYFMN huMov19 vHC CDR2 SEQ ID NO: 2RIHPYDGDTFYNQKFQG huMov19 vHC CDR3 SEQ ID NO: 3 YDGSRAMDY huMov19 vHCSEQ ID NO: 4 QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDYWGQGTTVTVSShuMov19 vHC nucleic acid sequence SEQ ID NO: 5aagcttgccaccatgggttggtcatgcatcatcctcttcttggttgcaactgctaccggagtgcacagtcaggtacagctcgtgcagtccggcgccgaggtggtgaagcctggtgccagcgtgaagatctcctgtaaagccagtggatacacattcaccggttattttatgaattgggtgaaacagagcccaggccaatccctcgaatggatagggcgaatccacccatatgacggggacaccttttacaaccagaaattccaggggaaagccactctgacagtggacaagagttccaacactgcacacatggagcttctctccctgaccagcgaagacttcgctgtttattactgtacccgttatgatggttcccgtgcaatggactactggggccaagggaccactgtcaccgtaagttccgccagcaccaagggccchuMov19 HC amino acid sequence SEQ ID NO: 6QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKhuMov19 vLC CDR1 SEQ ID NO: 7 KASQSVSFAGTSLMH huMov19 vLC CDR2SEQ ID NO: 8 RASNLEA huMov19 vLC CDR3 SEQ ID NO: 9 QQSREYPYThuMov19 vLCv1.00 SEQ ID NO: 10DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREY PYTFGGGTKLEIKRhuMov19 vLCv1.60 SEQ ID NO: 11DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREY PYTFGGGTKLEIKRhuMov19 LCv1.00 SEQ ID NO: 12DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEChuMov19 LCv1.60 SEQ ID NO: 13DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEChuMovl9 LCv1.00 nucleic acid SEQ ID NO: 14gaattcgccaccatgggctggagctgcattatcctttactggtagccacagctacaggcgtgcatagcgatatcatgagacacaatcccccctctctctggccgtgtcactcggacagcccgctatcatcagctgcaaagccagccagtctgtcagcttcgaggaacaagtcttatgcattggtatcatcagaagcctggccagcaacccaggctgctgatctatcgagcctcaaacttggaagcaggagtgccagaccggtatctgggtccgggagtaattaccgatatacacttaatatctcacctgtcgaggccgaggacgccgccacctactactgtcagcagagccgagagtaccatacacttttggcggtgggactaaactggaaa taaaacgtacghuMov19 LCv1.60 nucleic acid SEQ ID NO: 15gaattcgccaccatgggctggtcttgtatcatcctgatctggtggccaccgcaaccggtgttcactccgacattgtgctgacacagtcccccattcactggctgtatccctcggccagcccgctatcatcagctgcaaggctagccagagcgtgagttttgccggcacttcacttatgcattggtaccatcagaaaccaggccagcaacctaggctgctgatttatcgggctagcaacctggaggccggcgtgcccgaccgattagcgggagcggctccaagactgacttcactctgaccatctcccccgtagaagcagaagatgctgcaacctactactgtcagcagtctcgcgagtatccttatacattcggaggcggaactaaactgga gattaaacgtacgmuMov19 vHC CDR2 SEQ ID NO: 16 RIHPYDGDTFYNQNFKD muMov19 vHC_CAA68252SEQ ID NO: 17 QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRSHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDYWGQGTTVTVSmuMov19 vLC_CAA68253 SEQ ID NO: 18DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREY PYTFGGGTKL chMov19 HCSEQ ID NO: 19 QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK chMov19 LCSEQ ID NO: 20 DIELTQSPASLAVSLGQRAIISCKASQSVSTAGTSIMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECchMov19 HC nucleic acid SEQ ID NO: 21aagcttgccaccatgggttggtcttgtattatcctctttctcgtcgcaaccgcaacaggcgtccattcacaagtccaactgcagcaatccggcgccgaactcgttaaacctggagcatctgttaaaatctcatgtaaagcatcaggatactcatttactggctattttatgaactgcgtcaaacaatcacacggaaaatcacttgaatggatcggacgtattcacccctatgatggcgatactttttacaaccagaacttcaaagacaaagctacactcaccgttgacaaatcatctaacaccgacacatggaactcctttcactcacatctgaagacttcgctgatattactgtactagatacgatggatcaagagctatggattattggggacaaggaacaacagtcacagtctcatctgcatcaactaagggccca chMov19 LC nucleic acidSEQ ID NO: 22 gaattcgccaccatgggaggtcttgtattatcctctactcgtcgcaaccgcaacaggcgtccattcagatatcgaactcacacaatcaccagatccctcgcagtctactcggtcaacgcgcaatcatctcttgtaaagcctcccaatcagtctcattcgccggcacgtccctcatgcattggtaccatcaaaaacccggtcagcaacccaaactccttatctatagagcaagcaacctcgaagcaggcgttcccaccagatttagcggatcaggaagtaaaaccgatttcacactcaacattcatccagtcgaagaagaagatgcagctacttattattgccaacagtctagagaatatccatacacattcggagggggtaccaaacttgaa attaaacgtacgmuMov19 vHC_CAA68252 SEQ ID NO: 23QVQLQQSGAELVKPGASVKISCKASGYSFFGYFMNWVKQSHGKSLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDYWGQGTTVTVSmuMov19 vLC_CAA68253 SEQ ID NO: 24DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREY PYTFGGGTKLhuman folate receptor 1 SEQ ID NO: 25MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFL LSLALMLLWLLShuman folate receptor 1 nucleic acid sequence SEQ ID NO: 26Atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggccaggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgaccctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccactgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgaatgctcccccaacttggggccctggatccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc FR1-21 vLC CDR1 SEQ ID NO: 27KASDHINNWLA FR1-21 vLC CDR2 SEQ ID NO: 28 GATSLET FR1-21 vLC CDR3SEQ ID NO: 29 QQYWSTPFT FR1-21 vHC CDR1 SEQ ID NO: 30 SSYGMSFR1-21 vHC CDR2 SEQ ID NO: 31 TISSGGSYTY FR1-21 vHC CDR3 SEQ ID NO: 32DGEGGLYAMDY FR1-21 Kabat murine CDR-H2 SEQ ID NO: 33 TISSGGSYTYYPDGVKGFR1-21 Kabat human CDR-H2 SEQ ID NO: 34 TISSGGSYTYYSPGFQG muFR1-21 vLCSEQ ID NO: 35 DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTF GSGTKLEIKRmuFR1-21 vHC SEQ ID NO: 36EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSYTYYPDGVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR DGEGGLYAMDYWGQGTSVTVSSmuFR1-21 vLC DNA sequence SEQ ID NO: 37gacatccagatgacacaatcttcatcctacttgtctgtatctctaggaggcagagtcaccattacttgcaaggcaagtgaccacataaataattgttagcctggtatcagcagaaaccaggaaagctcctaggctcttaatatctggtgcaaccagtttggaaactgggttccttcaagattcagtggcagtggatctggaaagattacactctcttcatttccagtcttcagactgaagatgagctacttattactgtcaacagtattggagtactccattcacgttcggctcggggacaaagttggaaataaaacg muFR1-21HCvarPat SEQ ID NO: 38gaagtgaagctggtggagtagggggagacttagtgaagcctggagggtccctgaaactacctgtgcagcctctggattcactttcagtagctatggcatgtcttgggttcgccagactccagacaagaggaggagtgtgtcgcaaccattagtagtggtggtagttacacctactatccagacggtgtgaaggggcgattcaccataccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtagaggacacagccatgtattactgtgcaagggacggcgaggggggcctctatgctatggactactggggtcaaggaacctcagtc accgtctcacamuFR1-21 LC SEQ ID NO: 39DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTFGSGTKLE1KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNECmuFR1-21 HC SEQ ID NO: 40EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSYTYYPDGVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDGEGGLYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVWDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLK NYYLKKTISRSPGKhuFR1-21 vLC SEQ ID NO: 41DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLEFGVPSRFSGSGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTF GQGTKLEIKRhuFR1-21 vHC SEQ ID NO: 42EVQLVESGGDVVKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSYTYYSPGEQGRFTISRDKSKNTLYLQMSSLKAEDTAMYYCAR DGEGGLYAMDYWGQGTSVTVSShuFR1-21 VH_co SEQ ID NO: 43aagcttgccaccatgggatggtcatgcatcattctttttctcgtcgccactgccacaggtgtgcattccgaggtgcaacttgtagaatctggcggggatgttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatctgggtttaccttctcttcctacggaatgagagggtgagacagactcctggcaaggggctggagtgcgttgccaccattagtagtggaggttcttacacctactattcacctggattcagggacgctttacaatctcccgcgataagtctaagaacaccattacctccagatgagtagccttaaggctgaggacacagccatgtattattgcgctcgcgatggggagggagggattacgctatggactactggggccagggtaccagcgtgaccgtttcctctgctagtaccaagggcc c huFR21 VL_coSEQ ID NO: 44 gaattcgccaccatgggatggtcatgtatcattctgacttggtagcaacagcaactggcgtccattctgacatccagatgacccaatcctccagcagcttgtcagtatccgttgggggccgcgttactattacctgtaaggcctccgaccatataaataactggcttgcatggtatcaacagaagcctgggaaggcacctaaactgcttatactggggccacaagcctggagaccggcgtgccaccaggttctctggaagtggatctggcaaggactataccttgagcattagtagccttcaacctgaggacgtcgccacctactattgtcagcagtattggtctacaccattacctaggacagggcactaaattggagataaaacgtacg huFR1-21 LCSEQ ID NO: 45 DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEChuFR1-21 HC SEQ ID NO: 46EVQLVESGGDWKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSYTYYSPGFQGRFTISRDKSKNTLYLQMSSIJCAEDTAMYYCARDGEGGLYAMDYWGQGTSVTVSSASTKGPSWPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGhuFR1-21 LC DNA sequence SEQ ID NO: 47gacatccagatgacccaatcctccagcagcttgtcagtatccgttgggggccgcgttactattacctgtaaggcctccgaccatataaataactggcttgcatggtatcaacagaagcctgggaaggcacctaaactgcttatctctggggccacaagcctggagaccggcgtgccttccaggttctctggaagtggatctggcaaggactataccttgagcattagtagccttcaacctgaggacgtcgccacctactattgtcagcagtattggtctacaccctttacctttggacagggcactaaattggagataaaacgtacggtggctgcaccatctgtatcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgagtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagag tgthuFR1-21 HC DNA sequence SEQ ID NO: 48gaggtgcaacttgtagaatctggcggggatgttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatctgggtttaccttctcttcctacggaatgagctgggtgagacagactcctggcaaggggaggagtgcgttgccaccattagtagtggaggttcttacacctactattcacctggattcagggacgattacaatctcccgcgataagtctaagaacaccattacctccagatgagtagccttaaggctgaggacacagccatgtattattgcgctcgcgatggggagggagggctttacgctatggactactggggccagggtaccagcgtgaccgtttcctctgctagtaccaagggcccatcagattccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttacccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtccatctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagacctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtacacttgtctggtcaaggggttttaccatctgacattgagtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttatcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcc cttagcccaggghuFo1R1 DNA sequence EcoRI to XbaI SEQ ID NO: 49gaattcgccaccatggcacagcgcatgaccactcagacctgatctgttggtttgggtggcagtcgtgggagaggcccagaccaggattgcttgggcacgcacagagctgcttaatgtttgcatgaacgcaaagcaccataaagagaaacccggtcccgaggataagttgcacgaacagtgccgcccttggagaaagaatgcatgctgtagcacgaacacctutcaggaggcgcataaagacgtaagctatttgtatagatttaactggaaccattgcggtgaaatggcacctgcctgtaaacggcactttatccaggatacttgcttgtacgagtgtagcccgaatctcgggccaggattcagcaagttgatcagagttggcgcaaagagagggtgctgaacgttccgctttgcaaggaggactgcgagcaatggtgggaagactgtagaaccagctacacctgtaagtctaactggcacaaaggatggaactggacatccgggtttaacaaatgcgctgtcggcgctgcctgccagccatttcatttctactttccaactcccactgtcctgtgtaacgagatttggacgcattcatataaagtcagcaactacagccggggaccggccgctgcattcagatgtggttcgaccctgcacagggcaaccctaacgaggaggtcgcacgcttctacgctgcagaatgtctggagccggtccttgggctgcttggccatttctccttagcctcgccctcatgcttctctggctgagtcataatcta ga Primer EcoMH1SEQ ID NO: 50 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC Primer EcoMH2SEQ ID NO: 51 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG Primer BamIgG1SEQ ID NO: 52 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC SacIMK SEQ ID NO: 53GGAGCTCGAYATTGTGMTSACMCARWCTMCA HindKL SEQ ID NO: 54TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGCMixed bases are defined as follows: N = G + A + T + C, S = G + C, Y =C + T, M = A + C, R = A + G, W = A + T cd37-1LClead SEQ ID NO: 55ttttgaattcgccaccatgaagtttccttctcaacttcthuman and chimeric Mov19 vHC CDR2 composite SEQ ID NO: 56RIHPYDGDTFYNQXaa₁FXaa₂Xaa₃ Xaa₁ = Q, H, K, or R Xaa₂ = R, Q, H, or NXaa₃ = E, T, S, G, A, or V FR1-48vL CDR1 SEQ ID NO: 57 RASENIYSNLAFR1-48vL CDR2 SEQ ID NO: 58 AATNLAD FR1-48vL CDR3 SEQ ID NO: 59QHFWASPYT FR1-48vH CDR1 SEQ ID NO: 60 TNYWMQ FR1-48vH CDR2 SEQ ID NO: 61AIYPGNGDSR FR1-48vH CDR3 SEQ ID NO: 62 RDGNYAAY FR1-49vL CDR1SEQ ID NO: 63 RASENIYTNLA FR1-49vL CDR2 SEQ ID NO: 64 TASNLADFR1-49vL CDR3 SEQ ID NO: 65 QHFWVSPYT FR1-49vH CDR1 SEQ ID NO: 66 TNYWMYFR1-49vH CDR2 SEQ ID NO: 67 AIYPGNSDTT FR1-49vH CDR3 SEQ ID NO: 68RHDVGAMDY FR1-57vL CDR1 SEQ ID NO: 69 RASQNINNNLH FR1-57vL CDR2SEQ ID NO: 70 YVSQSVS FR1-57vL CDR3 SEQ ID NO: 71 QQSNSWPHYTFR1-57vH CDR1 SEQ ID NO: 72 SSFGMH FR1-57vH CDR2 SEQ ID NO: 73YISSGSSTIS FR1-57vH CDR5 SEQ ID NO: 74 EAYGSSMEY FR1-65vL CDR1SEQ ID NO: 75 KASQNVGPNVA FR1-65vL CDR2 SEQ ID NO: 76 SASYRYSFR1-65vL CDR3 SEQ ID NO: 77 QQYNSYPYT FR1-65vH CDR1 SEQ ID NO: 78 TSYTMHFR1-65vH CDR2 SEQ ID NO: 79 YINPISGYTN FR1-65vH CDR3 SEQ ID NO: 80GGAYGRKPMDY muFR1-48 Kabat defined HC CDR2 SEQ ID NO: 81AIYPGNGDSRYTQKFKG huFR1-48 Kabat defined HC CDR2 SEQ ID NO: 82AIYPGNGDSRYTQKFQG muFR1-49 Kabat defined HC CDR2 SEQ ID NO: 130AIYPGNSDTTYNLKFKG huFR1-49 Kabat defined HC CDR2 SEQ ID NO: 83AIYPGNSDTTYNQKFQG muFR1-57 Kabat defined HC CDR2 SEQ ID NO: 84YISSGSSTISYADTVKG huFR1-57 Kabat defined HC CDR2 SEQ ID NO: 85YISSGSSTISYADSVKG muFR1-65 Kabat defined HC CDR2 SEQ ID NO: 86YINPISGYTNYNQKFKD huFR1-65 Kabat defined HC CDR2 SEQ ID NO: 87YINPISGYTNYNQKFQG muFR1-48vL SEQ ID NO: 88DIQMTQSPASLSVSVGEFVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSESGTQYSLKINSLQSEDEGSYYCQHFWASPYTF GGGTKLEIKR muFR1-48vHSEQ ID NO: 89 QVQLQQSGAELARPGASVKLSCRASGYTETNYWMQWIKQRPGQGLEWIGATYPGNODSRYTQKFKGKATLTADKSSSTAYNIQVSSLISEDSAVYYCA RRDGNYAAYWGQGTLVIVSAmuFR1-49vL SEQ ID NO: 90DIQMTQSPASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTF GGGTKLEIKR muFR1-49vHSEQ ID NO: 91 EVQLQQSGTVLARPGASVKIVISCKASGYKFTNYWMYWIKQRPOQGLELIGAIYPGNSDTTYNLKFKGKAKLTAVTSANYVYMEVSSLTNEDSAVYYCTKRHDYGAMIDYWGQGTSVTVSS muFR1-57vL SEQ ID NO: 92DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPHYT FGGGTKLEIKR muFR1-57vHSEQ ID NO: 93 DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTISYADTVKGRFTISRDNSKKTLLLQMTSLRSEDTAMYYCAR EAYGSSMEYWGQGTSVTVSSmuFR1-65vL SEQ ID NO: 94DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYSEVPDRFTGSGSGTDFTLTISNMQSADLAEYFCQQYNSYPYTF GGGTKLEIKR muFR1-65vHSEQ ID NO: 95 QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCAS GGAYGRKPMDYWGQGTSVTVSShuFR1-48vL SEQ ID NO: 96DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGVPSRFSGSESGTDYSLKINSLQPEDFGSYYCQHFWASPYTF GQGTKLEIKR huFR1-48vHSEQ ID NO: 97 QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYTQKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCAR RDGNYAAYWGQGTLVTVSAhuFR1-49vL SEQ ID NO: 98DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGVPSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHFWVSPYTF GQGTKLEIKR huFR1-49vHSEQ ID NO: 99 QVQLOQSGAVVAKPGASVKMSCXASGYTFTKYWMYWIKQRPGQGLELIGAIYPGNSDTTYNQKFQGKATLTAWSANTWMEVSSLTSEDSAVYYCTKRH DYGAMDYWGQGTSVTVSShuFR1-57vL SEQ ID NO: 100ETVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIPDRFSGSGSGTDFTLSISSVEPEDFGMYFCQQSNSWPHYT FGQGTKLEIKR huFR1-57vHSEQ ID NO: 101 EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTISYADSVKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAR EAYGSSMEYWGQGTLVTVSShuFR1-65vL SEQ ID NO: 102EIVMTQSPATMSTSPGDRVSVTCKASQNVGPNVAWYQQKPGQSPRALIYSASYRYSGVPARFTGSGSGTDFILTISNMQSEDLAEYFCQQYNSYPYTF GQGTKLEIKR huFR1-65vHSEQ ID NO: 103 QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPOQGLAWIGYINPISGYTNYNQKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYNCAS GGAYGRKPMDYWGQGTSVTVSSmuFR1-48LC SEQ ID NO: 104DIQMTOSPASLSVSVGETVTITCRASENIYSNLAWYQQKOGKSPQLLVYAATNLADGVPSRFSGSESGTOYSLKINSLQSEDFGSYYCQHFVVASPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDiNVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNECmuFR1-48HC SEQ ID NO: 105QVQLQQSGAELARPGASVKLSCRASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYTQKFKGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK muFR1-49LCSEQ ID NO: 106 DIQMTQSTASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTFGGQTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYTKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTGEA TEIKTSTSPIVKSFNRNECmuFR1-49HC SEQ ID NO: 107EVQLQQSGTVLARPGASVKIVISCKASGYKFTNYWMYWIKQRPGQGLELIGAIYPONSDITYNLKFKGKAKLIAVTSANTVYMEVSSLTNEDSAVYYCTKRHDYGAMDYWGQGTSVTVSSAKTTAPSYYPLAPVCGDTTOSSVTLGCLVKGYEPEPVTUFWNSGSLSSGVIEFFPANTQSDLYTESSSVTVISSTWPSQSFECNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVELFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQTSWEVNNVENIFITAQTQIHREDYNSTLRVVSAIPIQHQDWMSGKEEKCKVNNKDITAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVIDFMPEDIYVENVINNGKTEINYKINTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVITEGL INHHTTKSFSRTPGKmuFR1-57LC SEQ ID NO: 108DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPHYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERONGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNECmuFR1-57HC SEQ ID NO: 109DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTISYADTVKGRFTISRDNSKKTLLLQMTSLRSEDTAMYYCAREAYGSSMEYWGQGTSVWSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSF SRTPGK muFR1-65LCSEQ ID NO: 110 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYSEVPDRFTGSGSGTDFTLTISNMQSADLAEYFCQQYNSYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNECmuFR1-65HC SEQ ID NO: 111QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKJEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHFITEKSLSHS PGK huFR1-48LCSEQ ID NO: 112 DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGVPSRFSGSESGTDYSLKINSLQPEDFGSYYCQHFWASPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEChuFR1-48HC SEQ ID NO: 113QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNGDSRYTOKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSFIEDPEVKFNWYVDGVEVIINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG huFR1-49LCSEQ ID NO: 114 DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGVPSRESGSGSGTDYSLKINSLQPEDFGTYYCQHFWVSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEChuFR1-49HC SEQ ID NO: 115QVQLQQSGAVVAKPGASVKMSCKASGYTFTNYWMYWIKQRPGQGLELIGAIYPGNSDTTYNQKFQGKATLTAVTSANTVYMEVSSLTSEDSAVYYCTKRHDYGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG huFR1-57LCSEQ ID NO: 116 EIVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIPDRFSGSGSGTDFTLSISSVEPEDFGNINTCQQSNSWPHYTRIQGTKLEIKRTVAAPSVFIEPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEChuFR1-57HC SEQ ID NO: 117EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTISYADSVKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAREAYGSSMEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSMCALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG huFR1-65LCSEQ ID NO: 118 EIVMTQSPATMSTSPGDRVSVTCKASQNVGPNVAWYOQKPGQSPRALIYSASYRYSGVPARFTGSGSGTDFTLTISNMQSEDLAEYFCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEChuFR1-65HC SEQ ID NO: 119QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISGYTNYNQKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG huFR1-48_VLSEQ ID NO: 120 gaattcgccaccatgggatggagttgtatcatcctgtttatgtggctacagcacaggggtacactccgatattcaaatgacacagtccccttcatccctgtccgtcagtgtgggggaaagggttaccatcacctgccgtgcatcagagaaccactattccaacctcgcctggtaccaacagaaactggcaagtcccctaagctgttggtctacgccgctacaaacctcgccgatggggtgccttcccgtttcagtgggtcagagtcaggcaccgactattctctgaagatcaactccaccagcctgaggatttcggctcctattactgtcagcacttctgggctagtccatatactttcggccagggaaccaaacttgaaattaaacgtacg huFR1-48_VHSEQ ID NO: 121 aagcttgccaccatggggtggagctgcatcatccttatctggtggccactgccaccggcgtgcactctcaggtccaacttgtgcagagcggagccgaggtggccaaacccggagctagtgttaagactcatgtaaagcatctggctacacctttactaactactggatgcagtggatcaagcaacggccaggccagggcctggagtggattggtgctatttatcccggaaacggggatagcaggtacactcagaaatttcagggaaaggctacccttaccgccgataagagttcaccacagcatatatgcaagtctcctctctgacctcagaggatagtgctgtctattactgcgctcgccgggatggcaactatgcagcctattggggtcaaggcacccttgtgactgtatccgcagcaagcaccaagggccc huFR1-49_VL SEQ ID NO: 122gaattcgccaccatgggttggtcatgcattatcctgtttctggtcgcaacagcaacaggtgtgcacagtgacattcagatgacccaaagcccaccagtctgagcgtttccgtgggggaacgtgtcactatcacatgcagagcttccgagaatatttacactaacctcgcatggtaccagcagaaacccgggaagtctccaaaacttctcgtatatacagccagcaacttggcagatggggtgcccagccggtttagcggatctggttcaggcaccgactattctttgaaaattaattccctgcagcctgaggattttggtacctactattgccagcatttttgggtatcaccatacacttttggacagggaacaaagctggagatcaagcgt acg huFR1-49_VHSEQ ID NO: 123 aagcttgccaccatgggctggtcttgtattattctttttcttgtggccacagccacaggagtccattcacaggtacagctccaacagtctggcgcagttgtcgccaagcccggcgcctagtgaagatgagttgcaaggcctctggctacaccttcactaattattggatgtactggatcaaacattgccccggccagggtctggaactcattggagccatctacccaggcaactccgacacaacatacaatcagaagtttcagggcaaagcaaccctgaccgctgtaacctcagctaataccgtgtacatggaggtaagtagcttgactagtgaagattccgcagtatactattgcaccaagcgccatgattacggcgccatggattactggggccaaggtaccagtgtgaccgtgtcttccgcttccaccaagggccc huFR1-57_VLSEQ ID NO: 124 gaattcgccaccatgggctggtcatgcattattttgttcctggtcgccaccgcaaccggcgttcattccgaaattgttcttactcagagccctgcaaccttgagtgtgacacccggcgatcgggtctcactgagttgcagagcttcccagaatatcaacaataatctgcactggtatcagcagaagcctggccagtctcctcgcttgctgattaagtatgtctcacagagcgtgtcaggtatccctgaccgtttaccgggtcaggttcaggcaccgacttcacactgtccatttctagcgtggagcctgaggatttcggaatgtacttttgccagcagagcaatagctggcctcactacacctttggccaagggaccaagctggagatcaag cgtacg huFR1-57_VHSEQ ID NO: 125 aagcttgccaccatgggctggagagtatcatcttgttccttgtggccacagctactggcgtgcactccgaggtgcagctggtcgaatccggcggaggcctggtgcagcctggggggagtagacggctgtcctgcgctgcctctgggtttactttctcaagtacggtatgcactgggtgcgtcaggcccccgggaagggcctggaatgggttgcttatatatcatctggcagctccaccatttcttatgctgattccgttaagggacgcttcaccatttccagagacaacagtaagaaaacccactgctgcagatgacctctctccgcgccgaagacaccgcaatgtattattgtgctagagaggcctacggcagtagtatggaatactgggggcaggggaccctggtgaccgtgtcttccgcatctactaagggccc huFR1-65_VL SEQ ID NO: 126gaattcgccaccatgggctggtcttgcattattctgttcctggttgcaacagccactggcgtccattccgaaatcgtgatgacccaatctcccgccaccatgtctacctacccggggaccgggtgtctgtgacctgcaaggcctctcagaatgttggcccaaacgtggcatggtatcaacagaaaccagggcagtcacccagagccctgatttactccgcttcttacagatattcaggagttcccgcccggttcacaggtagtgggtccggcactgactttaccttgaccatttccaacatgcaatccgaggacctggccgaatacttctgtcagcagtacaattcatatccctatacattcggccaggggaccaagctggaaataaagcgt acg huFR1-65_VHSEQ ID NO: 127 Aagcttgccaccatgggctggtcatgcataatcctgttcctggtcgcaaccgctacaggtgtacactcccaggtgcagttggtgcagagcggggccgaagttgctaagcccggtgcaagtgtaaaaatgtcctgcaaagctagcgggtacacattcacatcctatactatgcattgggtaaaacagcgcccaggacaggggctcgcctggataggctatattaacccaatatcaggatacacaaactacaatcagaaatttcagggaaaggcaaccctgaccgccgacaagtcctcttctaccgcatatatgcagctcaactccctgaccagtgaagatagcgcagtgtattactgtgcctccggcggtgcttatggccggaaacccatggattactggggacaaggcacctccgtcacagtgagtagcgcctcaaccaag ggcccKabat Defined Mov19 HC CDR2 Murine SEQ ID NO: 128 RIHPYDGDTFYNQNFKDKabat Defined Mov19 HC CDR2 Human SEQ ID NO: 129 RIHPYDGDTFYNQKFQG

1-127. (canceled)
 128. An isolated polynucleotide comprising thenucleotide sequence of SEQ ID NO:5.
 129. The polynucleotide of claim128, wherein the polynucleotide further comprises the nucleotidesequence of SEQ ID NO:14 or SEQ ID NO:15.
 130. A vector comprising thepolynucleotide of claim
 128. 131. A vector comprising the polynucleotideof claim
 129. 132. A host cell comprising the polynucleotide of claim128.
 133. A host cell comprising the polynucleotide of claim
 129. 134.An isolated polynucleotide comprising the nucleotide sequence of SEQ IDNO:14 or SEQ ID NO:15.
 135. The polynucleotide of claim 134, wherein thepolynucleotide comprises the nucleotide sequence of SEQ ID NO:14. 136.The polynucleotide of claim 134, wherein the polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:15.
 137. A vector comprising thepolynucleotide of claim
 135. 138. A vector comprising the polynucleotideof claim
 136. 139. A host cell comprising the polynucleotide of claim135.
 140. A host cell comprising the polynucleotide of claim
 136. 141.An isolated polynucleotide encoding a polypeptide comprising a heavychain (HC) variable domain, wherein the HC variable domain comprises:(a) a HC CDR1 comprising the amino acid sequence of GYFMN (SEQ ID NO:1);(b) a HC CDR2 comprising the amino acid sequence of RIHPYDGDTFYNQKFQG(SEQ ID NO: 2); and (c) a HC CDR3 comprising the amino acid sequence ofYDGSRAMDY (SEQ ID NO:3).
 142. The isolated polynucleotide of claim 141,wherein the HC variable domain comprises the amino acid sequence of SEQID NO:4.
 143. The isolated polynucleotide of claim 142, wherein thepolypeptide comprising the HC variable domain comprises the amino acidsequence of SEQ ID NO:6.
 144. The isolated polynucleotide of claim 141,wherein the polynucleotide further encodes a polypeptide comprising alight chain (LC) variable domain, wherein the LC variable domaincomprises: (a) a LC CDR1 comprising the amino acid sequence ofKASQSVSFAGTSLMH (SEQ ID NO:7); (b) a LC CDR2 comprising the amino acidsequence of RASNLEA (SEQ ID NO:8); and (c) a LC CDR3 comprising theamino acid sequence of QQSREYPYT (SEQ ID NO:9).
 145. The isolatedpolynucleotide of claim 144, wherein the LC variable domain comprisesthe amino acid sequence of SEQ ID NO:10.
 146. The isolatedpolynucleotide of claim 145, wherein the polypeptide comprising the LCvariable domain comprises the amino acid sequence of SEQ ID NO:12. 147.The isolated polynucleotide of claim 144, wherein the LC variable domaincomprises the amino acid sequence of SEQ ID NO:11.
 148. The isolatedpolynucleotide of claim 147, wherein the polypeptide comprising the LCvariable domain comprises the amino acid sequence of SEQ ID NO:13. 149.The isolated polynucleotide of claim 144, wherein the HC variable domaincomprises the amino acid sequence of SEQ ID NO:4, and wherein the LCvariable domain comprises the amino acid sequence of SEQ ID NO:10. 150.The isolated polynucleotide of claim 149, wherein the polypeptidecomprising the HC variable domain comprises the amino acid sequence ofSEQ ID NO:6, and wherein the polypeptide comprising the LC variabledomain comprises the amino acid sequence of SEQ ID NO:12.
 151. Theisolated polynucleotide of claim 144, wherein the HC variable domaincomprises the amino acid sequence of SEQ ID NO:4, and wherein the LCvariable domain comprises the amino acid sequence of SEQ ID NO:11. 152.The isolated polynucleotide of claim 151, wherein the polypeptidecomprising the HC variable domain comprises the amino acid sequence ofSEQ ID NO:6, and wherein the polypeptide comprising the LC variabledomain comprises the amino acid sequence of SEQ ID NO:13.
 153. A vectorcomprising the polynucleotide of claim
 141. 154. A vector comprising thepolynucleotide of claim
 144. 155. A host cell comprising thepolynucleotide of claim
 141. 156. A host cell comprising thepolynucleotide of claim 144.